Antibodies targeting CD318 (CDCP1) and uses thereof

Abstract

CD318 (CDCP1) is a CUB-domain containing cell surface protein over-expressed in major types of cancers to promote tumor growth and metastasis. The present disclosure relates to the identification and characterization of novel anti-CD318 monoclonal antibodies. By targeting CD318 as a tumor associated antigen, the disclosure further relates to different modalities of using anti-CD318 antibodies to kill cancer cells, including CD318 antibody based immunocytokines, CD318 antibody-based immune modulators, and CD318 antibody drug conjugates.

Description

[0001] ANTIBODIES TARGETING CD318 (CDCP1) AND USES THEREOF Cross Reference to Related Application

[0002] [1] This application claims the priority to and benefits of U.S. Provisional Patent Application No. 63 / 567,023, filed on March 19, 2024, the contents of which are hereby incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] [2] This application contains a Sequence Listing electronically submitted as an XML file entitled “15271_0016_00304_SL” having a size of 155,527 bytes and created on March 13, 2025. The information contained in the Sequence Listing is incorporated herein by reference.

[0005] Field of Disclosure

[0006] [3] The present disclosure provides antibodies targeting CD318 (CDCP1) and applications of the antibodies in the forms of anti-CD318 antibody based immunocytokine, anti- CD318 antibody based immune modulators and anti-CD318 antibody conjugates.

[0007] Background of Disclosure

[0008] [4] CD318, also known as Cub Domain Containing Protein 1 (CDCP1), SIMA135, or Trask, is a single span transmembrane receptor with three CUB domains expressed on the surface of epithelial cells (1). The expression of CD318 is significantly upregulated in response to transformation by RAS (2). Overexpression of CD318 is detected in several major types of cancer, including breast, lung, pancreas, colorectum, kidney, ovary and prostate cancers (3, 4). The elevated expression of CD318 is associated with poor prognosis and overall survival of cancer patients (5-7). CD318 is an important mediator of aberrant cancer-associated cascades critical for survival, growth, metastasis, and treatment resistance of tumor cells. Besides, the activity of CD318 is regulated by limited proteolysis of its extracellular domain, converting the full length 135 kDa glycoprotein to a 70 kDa membrane spanning carboxyl-terminal fragment and an amino-terminal fragment with the CUB1 domain shed from the cancer cells (2, 8). Indeed, the shed N-terminal fragment of CD318 is a cancer biomarker that can be detected in the serum of cancer patients (9). In summary, CD318 is emerging as a potential biomarker and therapeutic target for a range of cancers.

[0009] [5] Considering CD318 as a tumor associated antigen, the present disclosure provides the identification and characterization of novel monoclonal antibodies against CD318 and the utilization of such antibodies in different modalities to treat CD318-associated diseases and conditions such as malignancies. Summary of Disclosure

[0010] [6] One aspect of the present disclosure relates to an anti-CD318 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising complementarity determining region (HCDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises SEQ ID NOs: 7, 13, or 19; the HCDR2 comprises SEQ ID NOs: 8, 14, or 20; and the HCDR3 comprises SEQ ID NOs: 9, 15, or 21; and / or comprising a light chain variable region comprising complementarity determining region (LCDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises SEQ ID NOs: 10, 16, or 22; the LCDR2 comprises SEQ ID NOs: 11, 17, or 23; and the LCDR3 comprises SEQ ID NOs: 12, 18, or 24.

[0011] [7] In some embodiments, the HCDR1, HCDR2, and HCDR3 respectively compnse: SEQ ID NOs: 7, 8, and 9; or SEQ ID NOs: 13, 14, and 15; or SEQ ID NOs: 19, 20, and 21; and the LCDR1, LCDR2, and LCDR3 respectively comprise: SEQ ID NOs: 10, 11, and 12; or SEQ ID NOs: 16, 17, and 18; or SEQ ID NOs: 22, 23, and 24.

[0012] [8] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 101, 104, 108 and 109 or comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 101, 104, 108, and 109; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 102, 103, 105, 106, 107, 110, and 111 or comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 102, 103, 105, 106, 107, 110, and 111.

[0013] [9] In some embodiments, the anti-CD318 antibody or antigen-binding fragment comprises heavy chain variable region SEQ ID NO: 101 and light chain variable region SEQ ID NO: 102; heavy chain variable region SEQ ID NO: 101 and light chain variable region SEQ ID

[0014] NO: 103; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID

[0015] NO: 105; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID

[0016] NO: 106; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID

[0017] NO: 107; heavy chain variable region SEQ ID NO: 108 and light chain variable region SEQ ID

[0018] NO: 110; heavy chain variable region SEQ ID NO: 108 and light chain variable region SEQ ID

[0019] NO: 111; heavy chain variable region SEQ ID NO: 109 and light chain variable region SEQ ID

[0020] NO: 110; or heavy chain variable region SEQ ID NO: 109 and light chain variable region SEQ ID NO: 111.

[0021]

[0010] In some embodiments, the anti-CD318 antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 201, 204, 208, 209, 212, 213, and 214, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NO: 201, 204, 208, 209, 212, 213, and 214, and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211.

[0022]

[0011] A further aspect of the present disclosure relates to an immunocytokine comprising an anti-CD318 antibody or antigen-binding fragment as disclosed herein and a native or attenuated cytokine (e.g., a native or attenuated IFNa).

[0023]

[0012] In some embodiments, the native or attenuated IFNa comprises an amino acid sequence of SEQ ID NO: 321, 322, 323, or 324.

[0024]

[0013] In some embodiments, the immunocytokine comprises a heavy chain comprising an ammo acid sequence selected from SEQ ID NOs: 301-312, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NO: 301-312, and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211.

[0025]

[0014] In some embodiments, the immunocytokine is capable of directing native or attenuated IFNa to cells expressing CD318 and inducing the death of the cells directly and / or stimulating immune cells to eliminate the cells.

[0026]

[0015] Another aspect of the present disclosure relates to a multispecific antibody, or antigen-binding fragment thereof, comprising: an anti-CD318 moiety that targets CD318, an anti-VEGF moiety that targets VEGF, and an anti-PD-Ll moiety that targets PD-L1, wherein the anti-CD318 moiety comprises an anti-CD318 antibody or antigen-binding fragment thereof as disclosed herein.

[0027]

[0016] In some embodiments, the multispecific antibody or antigen-binding fragment thereof comprises one or more (e.g., one, two, three, or four) anti-PD-Ll moiety that is an anti- PD-Ll single chain Fv (scFv).

[0028]

[0017] In some embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises an Fc region.

[0029]

[0018] In some embodiments, the Fc region comprises a first Fc chain and a second Fc chain, and wherein the heavy chain portion of the anti-CD318 moiety is connected to the N- terminus of the first Fc chain, the heavy chain portion of the anti-VEGF moiety is connected to the N-terminus of the second Fc chain, and the one or more anti-PD-Ll moiety is connected to the C-terminus of the first Fc chain and / or the second Fc chain.

[0030]

[0019] In some embodiments, the anti-VEGF moiety comprises: a heavy chain variable region comprising an amino acid selected from SEQ ID NOs: 411 and 413, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 411 and 413, and a light chain variable region comprising an amino acid selected from SEQ ID NOs: 412 and 414, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 412 and 414.

[0031]

[0020] In some embodiments, the anti-PD-Ll scFv comprises an amino acid sequence selected from SEQ ID NOs: 421 and 422, or comprises an amino acid sequence having at least 85% identity7to any one of SEQ ID NOs: 421 and 422.

[0032]

[0021] In some embodiments, the multispecific antibody or antigen-binding fragment comprises: (i) a first heavy chain fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 401, 404, and 405, (ii) a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211, (iii) a second heavy chain fusion protein comprising an amino acid sequence SEQ ID NO: 402, and (iv) a light chain comprising an amino acid sequence SEQ ID NO: 403.

[0033]

[0022] In some embodiments, the multispecific antibody or antigen-binding fragment is capable of neutralizing VEGF and blocking PD-L1 activity7around CD318-expressing cells where the anti-CD318 moiety binds to the CD318-expressing cells.

[0034]

[0023] A further aspect of the present disclosure relates to an antibody conjugate comprising an antibody moiety conjugated to a payload, wherein the antibody moiety is the anti- CD318 antibody or antigen-binding fragment thereof as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein.

[0035]

[0024] In some embodiments, the payload is a cytotoxic drug.

[0036]

[0025] In some embodiments, the antibody moiety is conjugated to the pay load through a cleavable or non-cleavable chemical linker.

[0037]

[0026] In some embodiments, the antibody conjugate is capable of killing tumor cells by targeted delivery of the cytotoxic drug payload into CD318-expressing tumor cells.

[0038]

[0027] In some embodiments, the payload is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0039]

[0028] An aspect of the present disclosure relates to a nucleic acid encoding one or more of the polypeptides of the anti-CD318 antibody or antigen-binding fragment thereof as disclosed herein, the immunocytokine as disclosed herein, or the multispecific antibody or antigen-binding fragment as disclosed herein.

[0040]

[0029] One aspect of the present disclosure relates to a vector comprising the nucleic acid disclosed herein. Another aspect of the present disclosure relates to a cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

[0030] A further aspect of the present disclosure relates to a method for preparing the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, or the multispecific antibody or antigen-binding fragment as disclosed herein, comprising: culturing the cell as disclosed herein, and obtaining the antibody or antigen-binding fragment; immunocytokine; or multispecific antibody or antigen-binding fragment from the culture, optionally using controlled Fab arm exchange of culture supernatants.

[0041]

[0031] Another aspect of the present disclosure relates to a method of preparing the antibody conjugate as disclosed herein, comprising conjugation of a pay load onto the anti- CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, or the multispecific antibody or antigen-binding fragment as disclosed herein, through a cleavable or non-cleavable chemical linker.

[0042]

[0032] An aspect of the present disclosure relates to a pharmaceutical composition comprising the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, or the antibody conjugate as disclosed herein, and a pharmaceutically acceptable carrier.

[0043]

[0033] A further aspect of the present disclosure relates to a method for treating or preventing a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, the antibody conjugate as disclosed herein, or the pharmaceutical composition as disclosed herein.

[0044]

[0034] In some embodiments, the cancer is selected from breast cancer, bladder cancer, colon cancer, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, and any cancer with CD318 expression.

[0045]

[0035] In some embodiments, the method for treating or preventing a cancer further comprises administering chemotherapy to the subject.

[0046]

[0036] A further aspect of the present disclosure relates to a method for treating or preventing a disease or condition associated with overexpression of CD318 in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, the antibody conjugate as disclosed herein, or the pharmaceutical composition as disclosed herein.

[0047]

[0037] A further aspect of the present disclosure relates to a method for inhibiting expression of CD318 in a cell, comprising contacting the cell with the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, the antibody conjugate as disclosed herein, or the pharmaceutical composition as disclosed herein.

[0048]

[0038] These and other embodiments of the present disclosure will be described in greater detail herein.

[0049] Brief Description of the Drawings

[0050]

[0039] Figure 1. Anti-CD318 antibody binding to full-length CD318. ELISA binding assays showing mouse anti -human CD318 antibodies CD318-9A2, CD318-3 Al l and CD318-6662 binding to full-length human CD318 (1A) and mouse CD318 (IB). The Y axes units are in Absorbance values at 450 nm. The x axes units are the concentration of the respective test articles in ng / mL units.

[0051]

[0040] Figure 2. Anti-CD318 antibody binding to truncated CD318. ELISA binding assays showing binding to human CD318 isoform with 30-343 amino acids by mouse anti-human CD318 antibodies CD318-9A2 and CD318-3 Al l (2A) and CD318-6662 (2B). The Y axes units are in Absorbance values at 450 nm. The x axes units are the concentration of the respective test articles in ng / mL units.

[0052]

[0041] Figure 3. Anti-CD318 antibody binding to CD318-expressing cells Flow cytometry based binding assays showing mouse anti -human CD318 antibodies CD318-9A2, CD318-3A11 and CD318-6662 binding to breast cancer cell line T-47D (3A) and pancreatic cancer cell line BxPC-3 (3B). The Y axes units are Mean Fluorescence Intensity (MFI). The x axes units are the concentration of the respective test articles in ng / mL units.

[0053]

[0042] Figure 4. Humanized anti-CD318 antibody binding to full-length CD318 ELISA binding assays showing binding to human CD318 by humanized CD318-9A2 (4A), humanized CD318-3A11 (4B), and humanized CD318-6662 (4C) antibodies. The Y axes units are in Absorbance values at 450 nm. The x axes units are the concentration of the respective test articles in ng / mL units.

[0054]

[0043] Figure 5. Humanized CD318 antibody binding to CD318-expressing cells. Flow cytometry -based cell binding assays showing binding to breast cancer cell line T-47D by variants of humanized CD318-9A2 (5A), humanized CD318-3 Al l (5B), and humanized CD318- 6662 (5C) antibodies. The Y axes units are Mean Fluorescence Intensity (MFI). The x axes units are the concentration of the respective test articles in ng / mL units.

[0055]

[0044] Figure 6. Expression of CD318 in tumor cell lines. Flow cytometry assays showing the expression of human CD318 in exemplary breast cancer cell lines (6A), pancreatic cancer cell lines (6B), lung cancer cell lines (6C), esophagus cancer cell lines (6D), gastric cancer cell lines (6E), and colon cancer cell lines (6F). In some cases, the expression levels of HER2 were also shown. The Y axes units are normalized Mean Fluorescence Intensity (MFI) with a fold of signal change over that of isotype control antibody .

[0056]

[0045] Figure 7. IFNa reporter assays for humanized CD318 antibody-IFNa fusion. HEK-Blue IFNa reporter assays showing the activation of SEAP reporter gene expression by exemplary IFNa fusions with humanized CD318-9A2 antibody (7A), CD318- 3A11 antibody (7B) and CD318-6662 antibody (7C). The Y axes units are in Absorbance values at 650 nm. The x axes units are the concentration of the respective test articles in pM units.

[0057]

[0046] Figure 8. Cytotoxicity assays for humanized CD318 antibody-IFNa fusion. Cytotoxicity assays showing the inhibition of proliferation of HCC827 cells by exemplary' IFNa fusions with humanized CD3I8-9A2 antibody (8A) and CD318-3A11 antibody (8B), and the inhibition of proliferation of BxPC-3 cells by exemplary IFNa fusions with humanized CD318- 9A2 antibody (8C) and CD318-3A11 antibody (8D). IFNa fusion with an anti-gpl20 antibody was also included as a null control. The Y axes units are percentage of cell proliferation relative to untreated cells. The X axes units are the concentration of the respective test articles in pM units.

[0058]

[0047] Figure 9. PBMC cytotoxicity assays for humanized CD318 antibody-IFNa fusion. Flow cytometry-based PBMC-mediated cytotoxicity assays showing exemplary IFNa fusions with humanized CD3I8-9A2 antibody and control antibodies in mediating PBMC cytotoxicity against CD318 expressing BxPC-3 cells in vitro. The Y axes units are percentage of BxPC-3 cell lysis. The X axes units are increasing ratios of effector cell to target cells.

[0059]

[0048] Figure 10. In vivo studies of humanized CD318 antibody-IFNa fusion. Humanized xenograft BxPC-3 tumor model showing the tumor growth inhibition efficacy of exemplary IFNa fusions with humanized CD3I8-9A2 antibody in the treatment of BxPC-3 tumors along with human donor PBMC. Figure 10A shows tumor volume upon treatment (n=5). The Y axes units are tumor volume. The X axes units are days post treatment. Figure 10B shows mice body weight changes upon treatment. The Y axes units are percentage changes in body weight. The X axes units are days post treatment.

[0060]

[0049] Figure 11. In vitro functional assays for anti-CD318, VEGF and PD-L1 multispecific antibody. Functional evaluation of anti-CD318, VEGF and PD-L1 multispecific antibody by CD318 binding assay, VEGF reporter assay, and PD1-PDL1 blockade reporter assay. Figure 11A. Flow cytometry-based binding assay showing anti-CD318, VEGF and PD- L1 multispecific antibody CD318-9A2VHhumlVLhuml x VEGF_IgGl_PDL1996-ScFv (abbreviated as CD318-9A2 x VEGF x PDLlScFv) and CD318-9A2VHhumlVLhuml_IgGl (abbreviated as CD318-9A2) antibody concentration-dependently binding to CD318 expressing BxPC-3 cells. The Y axes units are Mean Fluorescence Intensity (MFI). The X axes units are the concentration of the respective test articles in ng / mL units. Figure 11B. VEGF reporter assay showing the inhibition of VEGF -mediated reporter gene activation by exemplary CD318-9A2 x VEGF x PDLlScFv multispecific antibody CD318-9A2VHhumlVLhuml x VEGF_IgGl_PDL1996-ScFv (abbreviated as CD318-9A2 x VEGF x PDLlScFv) and anti- VEGF antibody. The Y axes units are percentage of maximum reporter gene activation driven by 25 ng / mL VEGF. The x axes units are the concentration of the respective test articles in ng / mL units. Figure 11C. PD1-PDL1 blockade reporter assay showing the activation of reporter gene activation by exemplary CD318-9A2 x VEGF x PDLlScFv multispecific antibody CD318- 9A2VHhumlVLhuml x VEGF_IgGl_PDL1996-ScFv (abbreviated as CD318-9A2 x VEGF x PDLlScFv) and anti-PD-Ll antibody Atezolizumab. The Y axes units are luminescence. The x axes units are the concentration of the respective test articles in ng / mL units.

[0061]

[0050] Figure 12. In vivo study of anti-CD318, VEGF and PD-L1 multispecific antibody. Humanized xenograft BxPC-3 tumor model showing the tumor grow th inhibition efficacy of exemplary anti-CD318, VEGF and PD-L1 multispecific antibody CD318- 9A2VHhumlVLhuml x VEGF_IgGl_PDL1996-ScFv (abbreviated as CD318-9A2 x VEGF x PDLlScFv) in the treatment of BxPC-3 tumors along with human donor PBMC. Figure 12A shows tumor volume upon treatment (n=5). The Y axes units are tumor volume. The X axes units are days post treatment. Figure 12B shows mice body weight changes upon treatment. The Y axes units are percentage changes in body weight. The X axes units are days post treatment.

[0062]

[0051] Figure 13. SDS-PAGE analysis of the CD318-MMAF antibody conjugate molecules. Gel images showing protein bands of CD318, HER2 and null control antibodies unconjugated or conjugated with MMAF by SDS-PAGE analysis under reduced condition.

[0063]

[0052] Figure 14. Cytotoxicity assays of the CD318-6662 ADC and CD318- 3A11_ADC. Cytotoxicity assays showing concentration-dependent cytotoxicity of CD318- expressing BxPC-3 cells (14A), HCC827 cells (14B), AGS cells (14C), SNU-5 cells (14D), HCT116 cells (14E), LS174T cells (14F), HT-29 cells (14G), PC-3 cells (14H), and HCC70 cells (141) by MMAF conjugated CD318, HER2 and null control antibodies. The Y axes units are Relative Light Unit (RLU). The X axes units are the concentration of the respective test articles in ng / mL units. (14 J). Cytotoxicity assay showing the concentration-dependent cytotoxicity of MDA-MB-231 cells by MMAE conjugated CD318-3A11 and null control antibodies and DS-8201. The Y axes units are percentage of cell viability. The X axes units are the concentration of the respective test articles in nM units.

[0053] Figure 15. Cytotoxicity assays of the CD318-9A2 MMAF and CD318- 6662_MMAF. Cytotoxicity assays showing the concentration-dependent cytotoxicity of CD318- expressing HCT116 cells (15A) and HCC827 cells (15B) by MMAF conjugated CD318-9A2, CD318-6662, and null control antibodies. The Y axes units are percentage of cell viability relative to the case without antibody added. The X axes units are the concentration of the respective test articles in nM units (n=2).

[0064]

[0054] Figure 16. Cytotoxicity assays of the CD318-6662 MMAF with different types of IgGl Fc. Cytotoxicity assays showing the concentration-dependent cytotoxicity of CD318-expressing BxPC-3 cells (16A), HCC827 cells (16B), and HCT116 cells (16C) by CD318-6662_MMAF molecules with native IgGl Fc or IgGl Fc with L234A / L235A mutations (IgGl AA), and null control antibodies. The Y axes units are percentage of cell viability relative to the case without antibody added. The X axes units are the concentration of the respective test articles in nM units (n=2).

[0065]

[0055] Figure 17. HIC and SEC chromatographic analysis of the CD318-MMAE molecules. Hydrophobic interaction chromatography (HIC) profile and statistics of CD318- 9A2_MMAE (17A) and CD318-3A11_MMAE (17C) for DAR estimations. Size exclusion chromatography (SEC) profile and statistics of CD318-9A2_MMAE (17B) and CD318- 3A11_MMAE (17D).

[0066]

[0056] Figure 18. Presence of CD318 in xenograft tumors. Immunohistochemistry (IHC) staining showing the expression of human CD318 in exemplary human cancer cell xenograft tumor slice samples.

[0067]

[0057] Figure 19. Xenograft tumor model study of the CD318-ADC. Human cancer cell xenograft tumor models showing the tumor growth inhibition efficacy of CD318- 9A2_MMAE and CD318-3A11_MMAE along with a null control antibody with MMAE (null_MMAE) and DS-8201 in BxPC-3 (19A), MDA-MB-231 (19C), HCC827 (19E) and RKO (19G) models. The Y axes units are tumor volume. The X axes units are days post treatment. Arrows marked the days of dosing. Figures 19B, 19D, 19F and 19H showed mice body weight changes upon treatment in corresponding models. The Y axes units are either percentage changes in body weight or body weights. The X axes units are days post treatment.

[0068]

[0058] Figure 20. Toxicity study of the CD318- ADC. Mice were dosed with PBS, 17.3 mg / kg null_MMAE or CD318-9A2_MMAE and the toxicity studies showing the treatment effects on body weight change (20A), alanine aminotransferase (ALT) level (20B), aspartate aminotransferase (AST) level (20C), serum creatinine (CREA-S) level (20D), Creatine kinase- MB (CK-MB) level (20E), white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), monocyte (Mon) or eosinophil (Eos) numbers (20F), red blood cell (RBC) numbers (20G), hemoglobin (HGB) level (20H), and platelet (PLT) count (201).

[0069]

[0059] Figure 21. Schematic drawings of CD318 x VEGF x PD-L1 multispecific antibodies. The multispecific antibodies illustrated in this figure have two different sets of heavy chain and light chain shown with different shading: one set constitutes a CD318 binding arm and another set constitutes a VEGF binding arm. The PD-L1 ScFv domain is shown as a shaded circle attached at the C-terminus of one or both heavy chains of CD318 binding arm and VEGF binding arm.

[0070] Detailed description

[0071] Definitions

[0072]

[0060] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth. If certain content of a reference cited herein contradicts or is inconsistent with the present disclosure, the present disclosure controls.

[0073]

[0061] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0074]

[0062] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing the present disclosure, exemplary materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

[0075]

[0063] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0076]

[0064] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, multi-specific (e.g., bispecific or trispecific) antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.

[0077]

[0065] “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and Cm). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The Vn and the VL regions may be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each Vn and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0078]

[0066] “Complementarity determining regions (CDR)” are “antigen binding sites” in an antibody. CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (10) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) “Hypervariable regions,” “HVR,” or “HV,” three in the Vn (Hl, H2, H3) and three in the VL (LI, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (11). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations are described (12). The term “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia or IMGT, unless otherwise explicitly stated in the specification.

[0079]

[0067] Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAi, IgA2, IgGi, IgCh, IgG? and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant regions.

[0080]

[0068] “Antibody fragments” or “antigen binding fragments” refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (Vn), or a light chain variable region (VL). Antibody fragments include well known Fab, F(ab’)2, Fa and Fvfragments as well as domain antibodies (dAb) consisting of one Vn domain. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains may pair intramolecularly, or intermol ecularly in those cases when the Vn and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Disclosure Publ. Nos. W01998 / 44001, WO1988 / 01649, WO1994 / 13804 and W01992 / 01047. It is known that antigen binding fragments exhibit antigen binding activity. For example, Fv fragments, consisting only of the V-domains of the heavy and light chains associated with each other may be monovalent for antigen binding. Smaller fragments such as individual V-domains (domain antibodies or dABs) and individual CDR's have also been shown to retain the binding characteristics of the parent antibody. Single chain variable fragment (scFv) constructs comprise a VH and a VL domain of an antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker (e.g., with a length of more than 12 amino acids), that forces intramolecular interaction, allowing self-assembly of the two domains into a functional epitope binding site. These small proteins (e.g., with a MW around 25000 Da) generally retain specificity and affinity for their antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules.

[0081]

[0069] “Monoclonal antibody” refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies typically bind one antigenic epitope, except that multispecific monoclonal antibodies bind multiple distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent.

[0082]

[0070] “Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities. “Isolated antibody” encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0083]

[0071] “Humanized antibody” refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

[0084]

[0072] “Human antibody” refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin. If the antibody contains a constant region or a portion of the constant region, the constant region also is derived from sequences of human origin.

[0085]

[0073] “Immunocytokine” refers to an antibody fusion protein that combines a cytokine (a protein involved in cell signaling in the immune system) with an antibody or antibody fragment. This fusion allows a targeted delivery of the cytokine to specific cells or tissues, enhancing the immune response against diseases such as cancer. By bringing the immune- activating effects of cytokines directly to the diseased cells, immunocytokines can improve the effectiveness and / or reduce the side effects of traditional cytokine therapies.

[0086]

[0074] “Antibody conjugate” refers to antibody or antibody fragment conjugated with a payload through a linker. “Antibody-drug conjugate” (ADC) refers to antibody or antibody fragment conjugated with a cytotoxic drug pay load through a linker. The antibody specifically targets cancer cells, delivering the toxic drug directly to them, which helps to minimize damage to healthy cells. This targeted approach improves the effectiveness of the treatment and / or reduces side effects compared to traditional chemotherapy.

[0075] “Anti-target” refers to an antibody or antibody domain that can bind to the specified target molecule such as CD318 (i.e., anti-CD318 is an antibody or antibody domain that can bind to CD318). The style “CD318” refers to a CD318 protein or CD318 gene product.

[0087]

[0076] The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of

[0088] Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

[0089]

[0077] Conventional one and three-letter amino acid codes are used herein as shown in

[0090] Table 1.

[0091] Table 1

[0092]

[0078] The polypeptides, nucleic acids, fusion proteins, and other compositions provided herein may encompass polypeptides, nucleic acids, fusion proteins, and the like that have a recited percent identity to an amino acid sequence or DNA sequence provided herein. The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity,” “percent homology,” “sequence identity,” or “sequence homology” and the like mean the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (an algorithm). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A. M., ed ), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences.

[0093]

[0079] The constant region sequences of the mammalian IgG heavy chain are designated in sequence as Cni-hinge-Cm-Cm. The “hinge,” “hinge region” or “hinge domain” of an IgG is generally defined as including Glu216 and terminating at Pro230 of human IgGi according to the EU Index but functionally, the flexible portion of the chain may be considered to include additional residues termed the upper hinge region referred to as residues Glu216 to Gly237 and the lower hinge referred to as residues Glu233 to Ser239 of the Fcregion where FcyR binding was generally attributed. Hinge regions of other IgG isotypes may be aligned with the IgGi sequence by placing the first and last cysteine residues forming inter dieavy chain S-S bonds. Although boundaries may vary slightly, as numbered according to the EU Index, the CHI domain is adjacent to the Vn domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and includes the first (most amino terminal) constant region of an immunoglobulin heavy chain, e.g., from about EU positions 118-215. The Fcdomain extends from amino acid 231 to amino acid 447; the CH2 domain is from about Ala231 to Lys340 or Gly341; and the CHS from about Gly341 or Gln342 to Lys447. The residues of the IgG heavy chain constant region of the CHI region typically terminate at Lys. An Fcdomain containing molecule may comprise at least the CH2 and the CHS domains of an antibody constant region, e.g., comprising at least a region from about Ala231 to Lys447 of IgG heavy chain constant region. An Fcdomain containing molecule may optionally comprise at least a portion of the hinge region.

[0094]

[0080] “Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For a discontinuous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” typically depends on the methodology used to identify the epitope.

[0095]

[0081] A “leader sequence” as used herein includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well- known in the art.

[0096]

[0082] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms also include polypeptides that have co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.

[0097]

[0083] Furthermore, as used herein, a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.

[0098]

[0084] The term “recombinant,” as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or sy nthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term “recombinant,” as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression from a recombinant polynucleotide. The term “recombinant,” as used with respect to a host cell or a virus, refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to a material (e.g., a cell, a nucleic acid, a protein, or a vector) that has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0099]

[0085] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to include a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.

[0100]

[0086] “Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotide may be DNA or RNA molecules, cDNA, or a hybrid of these, single stranded or double stranded.

[0101]

[0087] “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0102]

[0088] As used herein, the term “heterologous” used in reference to nucleic acid sequences, proteins or polypeptides, means that these molecules are not naturally occurring in the cell from which the heterologous nucleic acid sequence, protein or polypeptide was derived. For example, the nucleic acid sequence coding for a human polypeptide that is inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in that particular context. Whereas heterologous nucleic acids may be derived from different organism or animal species, such nucleic acid need not be derived from separate organism species to be heterologous. For example, in some instances, a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be heterologous to a cell into which it is introduced in that the cell did not previously contain the synthetic nucleic acid. As such, a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be considered heterologous to a human cell, e.g., even if one or more components of the synthetic nucleic acid sequence or a polypeptide encoded therefrom was originally derived from a human cell.

[0103]

[0089] A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic or prokaryotic cell or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, wherein the cell can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a genetically modified eukaryotic host cell is genetically modified by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0104]

[0090] “Specific binding” or “specifically binds” or “binds” or “targets” or “targeting” refer to an antibody binding to a specific antigen with greater affinity than for other antigens. Typically, the antibody “specifically binds” when the equilibrium dissociation constant (KD) for binding is about I xlO'8M or less, for example about I x lO'9M or less, about I xlO'10M or less, about 1 x 10'11M or less, or about 1 fiO’12M or less, typically with the KD that is at least one hundred-fold less than its KD for binding to a non-specific antigen (e.g., BSA, casein). The KD may be measured using standard procedures.

[0105]

[0091] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0106]

[0092] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g. , rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

[0107]

[0093] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0108]

[0094] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0109] Anti-CD318 antibody

[0110]

[0095] In some embodiments, the present disclosure provides antibodies targeting CD318 (also an “anti-CD318 antibody”) or antigen binding fragments thereof. The anti-CD318 antibodies encompass a full length antibody comprising two heavy chains and two light chains and antigen binding fragment thereof. In some embodiments, the antibodies are human or humanized antibodies. Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody vanable region linked to a human constant region. CDR-grafted antibodies are antibodies that include the CDRs from a non-human “donor” antibody linked to the framework region from a human “recipient” antibody. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. The present antibodies can be of any class (IgG, IgM, IgE, IgA, IgD, etc.) or isotype. For example, a human antibody can comprise an IgG Fc domain, such as at least one of the isotypes, IgGl, IgG2, IgG3 or IgG4.

[0111]

[0096] In some embodiments, the present disclosure provides an antibody targeting CD318 (e.g., the humanized antibody 9A2, the humanized antibody 3A11, or the humanized antibody 6662), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises SEQ ID NOs: 7, 13, or 19; the HCDR2 comprises SEQ ID NOs: 8, 14, or 20; and the HCDR3 comprises SEQ ID NOs: 9, 15, or 21; and / or a light chain variable region comprising complementarity determining region (LCDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises SEQ ID NOs: 10, 16, or 22; the LCDR2 comprises SEQ ID NOs: 11, 17, or 23; and the LCDR3 comprises SEQ ID NOs: 12, 18, or 24.

[0097] In some embodiments, the present disclosure provides an antibody targeting CD318 (e.g., the humanized antibody 9A2), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 respectively comprise SEQ ID NOs: 7, 8, and 9; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 respectively comprise SEQ ID NOs: 10, 11, and 12. In some embodiments, the antibody targeting CD318 (e.g., humanized antibody 9A2) comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 101 or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 101, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 102 and 103, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 102 and 103.

[0112]

[0098] In some embodiments, the present disclosure provides an antibody targeting CD318 (e.g., the humanized antibody 3 Al 1), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 13, 14, and 15, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, the humanized antibody 3 Al 1 targeting CD318 comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 104 or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 104, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 105, 106 and 107, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 105, 106 and 107.

[0113]

[0099] In some embodiments, the present disclosure provides an antibody targeting CD318 (e.g., the humanized antibody 6662), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 respectively comprise an amino acid sequence of SEQ ID NOs: 19, 20, and 21; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequence SEQ ID NOs: 22, 23, and 24. In some embodiment, the antibody targeting CD318 (e.g., humanized antibody 6662) comprises a heavy chain variable region comprising an amino acid selected from SEQ ID NOs: 108 and 109, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 108 and 109, and / or a light chain variable region comprising an amino acid sequence of selected from SEQ ID NOs: 110 and 111, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 110 and 111.

[0114]

[0100] In some embodiments, the anti-CD318 antibody disclosed herein (e.g., antibody 9A2) comprises an anti-CD318 antibody heavy chain variable region comprising an amino acid sequence and a IgGl Fc selected from SEQ ID NOs: 201 and 212, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 201 and 212, and an anti-CD318 antibody light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0115]

[0101] In some embodiments, the anti-CD318 antibody (e.g., antibody 3A11) disclosed herein comprises an anti-CD318 antibody heavy chain variable region comprising an amino acid sequence and a IgGl Fc selected from SEQ ID NOs: 204 and 213, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 204 and 213, and an anti-CD318 antibody light chain comprising an amino acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0116]

[0102] In some embodiments, the anti-CD318 antibody (e.g., antibody 6662) disclosed herein comprises an anti-CD318 antibody heavy chain variable region comprising an amino acid sequence and a IgGl Fc selected from SEQ ID NOs: 208, 209 and 214, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 208, 209 and 214, and an anti-CD318 antibody light chain comprising an amino acid sequence selected from SEQ ID NOs: 210 and 211, or comprising an ammo acid sequence having at least 85% (e g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 210 and 211. Anti-CD318 antibody based immunocytokines with an interferon

[0117]

[0103] In some embodiments, the present disclosure provides an immunocytokine comprising an anti-CD318 antibody or antigen binding fragment as disclosed herein and a native or attenuated cytokine such as interferon (also called an ”anti-CD318 based immunocytokine”). In some embodiments, the native or attenuated cytokine, such as an interferon, is fused at the C- terminus of the heavy chains of the anti-CD318 antibody disclosed herein. Without being bound by theory, in some embodiments, the binding of anti-CD318 antibody to cell surface CD318 antigen directs the cytokine (e.g., an interferon component) to the proximity of CD318- expressing cells, where the cytokine (e.g., interferon) enhances cytotoxicity of CD318- expressing cells directly and / or stimulates immune cells to eliminate CD318-expressing cells.

[0118]

[0104] In some embodiments, the anti-CD318 antibody based immunocytokine comprises native or attenuated type I interferon a (IFNa), type I interferon (IFNP), type II interferon y (IFNy), or type III interferon / . ( I FNZ).

[0119]

[0105] In some embodiments, the anti-CD318 antibody based immunocytokine comprises native or attenuated interferon al, a2, a4, a5, a6, a7, a8, alO, al3, al4, al6, al7, or a21.

[0120]

[0106] In some embodiments, the anti-CD318 antibody based immunocytokine comprises native or attenuated interferon a2a (IFNa2a) or interferon a2b (IFNa2b).

[0121]

[0107] In some embodiments, the anti-CD318 antibody based immunocytokine comprises native interferon a2b (IFNa2b) or attenuated IFNa2b with reduced IFNa2b activity.

[0122]

[0108] In some embodiments, the anti-CD318 antibody based immunocytokine comprises native interferon a2b (IFNa2b) comprising an amino acid sequence SEQ ID NO: 321.

[0123]

[0109] In some embodiments, the anti-CD318 antibody based immunocytokine comprises attenuated IFNa2b with optional mutations that attenuate the activity of IFNa2b. As a non-limiting example, the attenuated IFNa2b has an amino acid sequence comprising one or more mutations selected from L15A, M16A, R22A, R23A, L24A, S25A, L26A, F27A, F27S, L30A, L30S, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, R125A, K131A, E132A, K133A, K134A, R144A, R144I, R144T, A145D, A145G, A145H, A145M, M148A, R149A, R149K, S152A, L153A, L153K, and N156A substitutions, compared to SEQ ID NO: 321.

[0124]

[0110] In some embodiments, the anti-CD318 antibody based immunocytokine comprises attenuated IFNa2b with L15A mutation comprising an amino acid sequence SEQ ID NO: 322.

[0111] In some embodiments, the anti-CD318 antibody based immunocytokine comprises attenuated IFNa2b with L15A and L153K mutations comprising an amino acid sequence SEQ ID NO: 323.

[0125]

[0112] In some embodiments, the anti-CD318 antibody based immunocytokine comprises attenuated IFNoc2b with L30A mutation comprising an amino acid sequence SEQ ID NO: 324.

[0126]

[0113] In some embodiments, the anti-CD318 antibody based immunocytokine comprises a native or an attenuated IFNa2b fused at the C-terminus of one or both of the two heavy chains of the anti-CD318 antibody disclosed herein.

[0127]

[0114] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 9A2 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and native IFNa2b) as set forth as SEQ ID NO: 301, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 301, and an anti-CD318 antibody (e.g., 9A2) light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0128]

[0115] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 9A2 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L15A mutation) as set forth as SEQ ID NO: 302, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 302, and an anti-CD318 antibody (e.g., 9A2) light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0129]

[0116] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an amino acid sequence (e.g., an anti-CD318 antibody 9A2 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L15A and L153K mutations) as set forth as SEQ ID NO: 303, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 303, and an anti-CD318 antibody (e.g., 9A2) light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0130]

[0117] In some embodiments, the anti-CD318 antibody based immunocytokine comprises a heavy chain comprising an amino acid sequence (including e.g., an anti-CD318 antibody 9A2 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L30A mutation) as set forth as SEQ ID NO: 304, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 304, and an anti-CD318 antibody (e.g., 9A2) light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0131]

[0118] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an amino acid sequence (e.g., an anti-CD318 antibody 3A11 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and native IFNa2b) as set forth as SEQ ID NO: 305, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 305, and an anti-CD318 antibody (e.g., 3A11) light chain comprising an ammo acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0132]

[0119] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody

[0133] 3 Al 1 heavy chain variable region, an IgGl Fc with L234A / L235 A mutations, and IFNcc2b with L15A mutation) as set forth as SEQ ID NO: 306, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 306, and an anti-CD318 antibody (e.g., 3A11) light chain comprising an amino acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0134]

[0120] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody

[0135] 3 Al 1 heavy chain variable region, an IgGl Fc with L234A / L235 A mutations, and IFNa2b with L15A and L153K mutations) as set forth as SEQ ID NO: 307, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 307, and an anti-CD318 antibody (e.g., 3A11) light chain comprising an amino acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0136]

[0121] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody

[0137] 3 Al 1 heavy chain variable region, an IgGl Fc with L234A / L235 A mutations, and IFNcc2b with L30A mutation) as set forth as SEQ ID NO: 308, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 308, and an anti-CD318 antibody (e.g., 3A11) light chain comprising an amino acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0138]

[0122] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 6662 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and native IFNa2b) as set forth as SEQ ID NO: 309, or comprising an ammo acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 309, and an anti-CD318 antibody (e.g., 6662) light chain comprising an amino acid sequence selected from SEQ ID NOs: 210 and 211, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 210 and 211.

[0139]

[0123] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 6662 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L15A mutation) as set forth as SEQ ID NO: 310, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 310, and an anti-CD318 antibody (e.g., 6662) light chain comprising an amino acid sequence selected from SEQ ID NOs: 210 and 211, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 210 and 211.

[0140]

[0124] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 6662 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L15A and L153K mutations) as set forth as SEQ ID NO: 311, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 311, and an anti-CD318 antibody (e.g., 6662) light chain comprising an amino acid sequence selected from SEQ ID NOs: 210 and 211, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 210 and 211.

[0141]

[0125] In some embodiments, the anti-CD318 antibody based immunocytokine comprises aheavy chain comprising an ammo acid sequence (e.g., an anti-CD318 antibody 6662 heavy chain variable region, an IgGl Fc with L234A / L235A mutations, and IFNa2b with L30A mutation) as set forth as SEQ ID NO: 312, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 312, and an anti-CD318 antibody (e.g., 6662) light chain comprising an amino acid sequence selected from SEQ ID NOs: 210 and 211, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 210 and 211.

[0142] Multispecific antibodies targeting CD318, VEGF and PD-L1

[0143]

[0126] In some embodiments, the present disclosure provides a multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) based on the anti-CD318 antibody disclosed herein. Without being bound by theory, in some embodiments, the binding of anti-CD318 antibody to cell surface CD318 antigen localizes VEGF neutralization activity and PDL1-PD1 blockade from the CD318 x VEGF x PDL1 multispecific antibody at the CD318 expressing cells. In some embodiments, the VEGF neutralization activity blocks angiogenesis. In some embodiments, blockade of PD1-PD-L1 blockade activity stimulates T cells to eliminate CD318-expressing tumor cells.

[0144]

[0127] In some embodiments, the multispecific antibody, or antigen binding fragment thereof, comprises an anti-CD318 moiety that targets CD318, an anti -VEGF moiety that targets VEGF, and an anti-PD-Ll moiety that targets PD-L1, wherein the anti-CD318 moiety comprises the anti-CD318 antibody or antigen binding fragment as disclosed herein. The anti-CD318 moiety, anti-VEGF moiety, and anti-PD-Ll moiety can be in any form of “antibody fragments” or “antigen binding fragments” as described herein. In some embodiments, the anti-VEGF moiety comprises any anti-VEGF antibody or antigen binding fragments thereof known in the art. In some embodiments, anti-PD-Ll moiety comprises any anti-PD-Ll antibody or antigen binding fragments thereof known in the art.

[0145]

[0128] In some embodiments, the multispecific antibody comprises one or more anti- PD-Ll moiety that is an anti-PD-Ll single chain Fv (ScFv).

[0129] In some embodiments, the multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) comprises a first heavy chain and a first light chain that bind to CD318 (a CD318 binding arm), a second antibody heavy chain and a second light chain that binds to VEGF (a VEGF binding arm), and a single chain Fv (ScFv) that binds to PD-L1 fused at the C-terminus of one or both of the first and second heavy chains (as illustrated in Figure 21).

[0146]

[0130] In some embodiments, the antibody heavy chain and light chain that bind to VEGF comprises an anti-VEGF antibody bevacizumab heavy chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 411, and an anti-VEGF antibody bevacizumab light chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 412.

[0147]

[0131] In some embodiments, the antibody heavy chain and light chain that bind to VEGF comprises an anti-VEGF antibody heavy chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 413, and an anti-VEGF antibody light chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 414.

[0148]

[0132] In some embodiments, the single chain Fv (scFv) that binds to PD-L1 comprises a scFv domain derived from an anti-PD-Ll antibody atezolizumab Fab comprising an amino acid sequence as set forth as SEQ ID NO: 421.

[0149]

[0133] In some embodiments, the single chain Fv (scFv) domain that binds to PD-L1 comprises a ScFv domain derived from an anti-PD-Ll antibody 996 Fab comprising an amino acid sequence as set forth as SEQ ID NO: 422.

[0150]

[0134] In some embodiments, the multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) comprises a first heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 401 (e.g., (comprising an anti-CD318 antibody (e.g., 9A2) heavy chain variable region, an IgGl Fc with F405L mutation, and a ScFv domain derived from an anti-PD-Ll antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 401; an anti-CD318 antibody (e.g., 9A2) light chain comprising an amino acid sequence chosen from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203; a second heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 402 (e.g., comprising an anti-VEGF antibody heavy chain variable region, an IgGl Fc with K409R mutation, and a ScFv domain derived from an anti-PD-Ll antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 402; and an anti-VEGF antibody light chain comprising an amino acid sequence as set forth as SEQ ID NO: 403, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 403.

[0151]

[0135] In some embodiments, the multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) comprises a first heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 404 (e.g., comprising an anti-CD318 antibody 3A11 heavy chain variable region, an IgGl Fc with F405L mutation, and a scFv domain derived from an anti- PD-L1 antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 404; an anti-CD318 antibody 3 Al l light chain comprising an amino acid sequence chosen from SEQ ID NOs: 205, 206 and 207, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to any one of SEQ ID NOs: 205, 206 and 207; a second heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 402 (e.g., comprising an anti-VEGF antibody heavy chain variable region, an IgGl Fc with K409R mutation, and a scFv domain derived from an anti-PD-Ll antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 402; and an anti-VEGF antibody light chain comprising an amino acid sequence as set forth as SEQ ID NO: 403, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 403.

[0152]

[0136] In some embodiments, the multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) comprises a first heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 405 (e.g., comprising an anti-CD318 antibody (e.g., 6662) heavy chain variable region, an IgGl Fc with F405L mutation, and a scFv domain derived from an anti-PD-Ll antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 405; an anti-CD318 antibody 6662 light chain comprising an amino acid sequence chosen from SEQ ID NOs: 210 and 211, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to any one of SEQ ID NOs: 210 and 211; a second heavy chain comprising an amino acid sequence as set forth as SEQ ID NO: 402 (e.g., comprising an anti-VEGF antibody heavy chain variable region, an IgGl Fc with K409R mutation, and a ScFv domain derived from an anti-PD-Ll antibody 996 Fab), or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify' to SEQ ID NO: 402; and an anti-VEGF antibody light chain comprising an amino acid sequence as set forth as SEQ ID NO: 403, or comprising an amino acid sequence having at least 85% (e g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identify to SEQ ID NO: 403.

[0153]

[0137] In some embodiments, the multispecific antibody targeting CD318, VEGF and PD-L1 (CD318 x VEGF x PDL1) is generated by heterodimerization, through a controlled Fabarm exchange process, wherein a first parental antibody comprises anti-CD318 antibody heavy chains having an IgGl Fc with F405L mutation and a PD-L1 antibody Fab as ScFv fused at the C -terminus and anti-CD318 antibody light chains, and a second parental antibody comprises anti -VEGF antibody heavy chains having an IgGl Fc with K409R mutation and a PD-L1 antibody Fab as ScFv fused at the C-terminus and anti-VEGF antibody light chains.

[0154] Leader Sequences

[0155]

[0138] In some embodiments, a leader peptide drives the secretion of the antibody described in this disclosure into the cell culture supernatant as a secreted antibody protein. Any leader peptide for any known secreted proteins I peptides may be used.

[0156]

[0139] As used herein, a “leader peptide” or “signal peptide” or “leader sequence” refers to a short peptide, usually 16-30 amino acids in length, that is present at the N-terminus of most of newly synthesized proteins that are destined towards the secretory pathway. Lead peptides are extremely heterogeneous in sequence, and many prokaryotic and eukaryotic lead peptides are functionally interchangeable even between different species, and the efficiency of protein secretion may depend on the leader sequence. In some embodiments, the sequence of the leader I signal peptide is selected for high efficiency of protein secretion.

[0157]

[0140] In some embodiments, the leader peptide is a leader peptide from a protein residing either inside certain organelles (such as the endoplasmic reticulum, Golgi, or endosomes), secreted from the cell, or inserted into most cellular membranes.

[0158]

[0141] In some embodiments, the leader peptide is from a eukaryotic protein.

[0159]

[0142] In some embodiments, the leader peptide is from a secreted protein, e.g., a protein secreted outside a cell.

[0160]

[0143] In some embodiments, the leader peptide is from a transmembrane protein.

[0161]

[0144] In some embodiments, the leader peptide comprises a stretch of amino acids that is recognized and cleaved by a signal peptidase.

[0162]

[0145] In some embodiments, the leader peptide does not contain a cleavage recognition sequence of a signal peptidase.

[0163]

[0146] In some embodiments, the leader peptide is a signal peptide for tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSVgD), a growth hormone, a cytokine, a lipoprotein export signal, CD2, CD35, CD38, CD3y, CD3^, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR, or S. cerevisiae mating factor a-1 signal peptide.

[0164]

[0147] In some embodiments, a leader sequence as described herein is a mammalian CD4 or CD8 leader sequence, including but not limited to, e.g., a human CD4 or CD8 leader sequence, a non-human primate CD4 or CD8 leader sequence, a rodent CD4 or CD8 leader sequence, and the like. In some embodiments, a CD4 or CD8 leader comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the human CD4 or CD8 leader sequences.

[0165] Modifications to the IgG Fc region of anti-CD318 antibodies

[0166]

[0148] In some embodiments, the anti-CD318 antibodies disclosed herein comprise a modified IgG Fcregion, wherein the modified Fcregion comprises at least one amino acid modification relative to a native Fcregion, e.g., a native Fcregion from a parental antibody. In some embodiments, the anti-CD318 antibodies comprise a modified Fcregion that extends the half-life of the antibody as compared to a parental (or native) antibody. In some embodiments, the half-life of the antibody is the serum half-life or a half-life measured by an in vitro assay.

[0167]

[0149] Exemplary mutations that may be made singularly or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0168]

[0150] In some embodiments, the modified IgG Fcregion modified comprises M252Y / S254T / T256E mutations, collectively known as YTE mutations, in the IgGl Fcregion

[0169] (13).

[0170]

[0151] In some embodiments, the modified IgG Fc region comprises M428L / N434S mutations, collectively known as LS mutations, in the IgGi Fcregion, e.g., as in SEQ ID NO: 319

[0171] (14).

[0172]

[0152] In some embodiments, modified IgG Fcregion compnses T250Q / M428L mutations in the IgGi Fcregion (15).

[0173]

[0153] In some embodiments, the modified IgG Fcregion comprises a N434A mutation in the IgGi Fcregion (16).

[0174]

[0154] In some embodiments, the modified IgG Fc region comprises T307A / E380A / N434A mutations in the IgGi Fcregion (17).

[0175]

[0155] In some embodiments, the effect of Fcengineering on the extension of antibody half-life is evaluated in PK studies in mice relative to antibodies with a native (e.g., unmodified) IgG Fc region.

[0156] In some embodiments, the anti-CD318 antibodies disclosed herein comprise a modified Fcregion that enhances resistance to proteolytic degradation, e.g., by a protease that cleaves the naturally-occurring antibody between or at residues 222-237 (EU numbering), as compared to resistance to proteolytic degradation in a parental (or native) antibody.

[0176]

[0157] In some embodiments, the modified IgG Fcregion comprises E233P / L234A / L235A mutations in the hinge region with G236 deleted when compared to a native antibody, residue numbering according to the EU Index (18).

[0177]

[0158] Where effector functionality is to be enhanced, in some embodiments, the antibodies disclosed herein comprise a modified Fcregion, wherein at least one residue in the Fc region differs from the residue in a parental (or native) antibody, thereby increasing the binding of the antibody to an activating Fcy receptor (FcyR) and / or increasing Fceffector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP) as compared to the native antibody. Such modifications may comprise low or null Fc fucosy lati on, and / or engineering of Fc mutations such as S239E / I332E / A330L, S239D / I332E / A330L, S239D / I332E, S239D, I332E, S298A / E333A / K334A.

[0178]

[0159] In some embodiments, the modified Fcregion comprises F243L / R292P / Y300L / V305I / P396L mutations, collectively known as LPLIL mutations, as compared to a native IgG antibody, residue numbering according to the EU Index (19).

[0179]

[0160] In some embodiments, the modified Fc region comprises E345R or other Fc clustering mutations on the native IgG antibody using residue numbering according to the EU Index.

[0180]

[0161] In some embodiments, the modified Fc region comprises F243L / R292P / Y300E / V305I / P396L mutations and E345R mutation, collectively known as E345R / LPEIL mutations, when compared to a native IgG antibody, residue numbering according to the EU Index.

[0181]

[0162] In some embodiments, where effector functionality is not desired, the antibodies disclosed herein may be engineered to reduce effector functionality. In some embodiments, the antibodies comprise a modified Fcregion, wherein at least one residue in the Fcregion differs from a native IgG antibody, thereby reducing binding of the antibody to an activating Fcy receptor (FcyR) and / or reducing Fceffector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0163] Fc positions that may be mutated to reduce binding of the antibody to the activating FcyR and subsequently to reduce effector functions are those described for example in (20) (21) (22) (23) (16).

[0182]

[0164] Exemplary mutations made singularly or in combination comprise K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S mutations on IgGi, IgG , IgG? or IgG4.

[0183]

[0165] Exemplary combination mutations made to reduce ADCC comprise L234A / L235A on IgGi, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG , F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgGi, IgG?, IgG? or IgG4, V234A / G237A on IgG?, K214T / E233P / L234V / L235A / G236- deleted / A327G / P331A / D365E / L358M on IgGi, H268Q / V309L / A330S / P331S on IgG?, S267E / L328F on IgGi, L234F / L235E / D265A on IgGi, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgGi, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236-deleted / G237A / P238S on IgG4. In some embodiments, modified a modified Fcregions comprise hybrid IgGw Fcdomains, such as Fcwith residues 117-260 from IgG? and residues 261-447 from IgG4.

[0184]

[0166] In some embodiments, modified IgG Fcregion comprises L234A / L235A mutations, collectively known as AA mutations, compared to a parental native IgGi antibody Fc region, residue numbering according to the EU Index.

[0185]

[0167] In some embodiments, the modified IgG Fcregion comprises L234F / L235E / D265A mutations, collectively known as FEA mutations, compared to a parental native IgGi antibody Fc region, residue numbering according to the EU Index.

[0186]

[0168] In some embodiments, the modified IgG Fcregion comprises an IgG2 or IgG4 Fcregion instead of an IgGi Fcregion on the bispecific antibodies disclosed herein.

[0187]

[0169] In some embodiments, the antibodies disclosed herein comprise a modified Fcregion that facilitates generation of a bispecific antibody by Fc heterodimerization. In some embodiments, at least one residue in the modified Fcregion differs from a parental (or native) antibody.

[0188]

[0170] In some embodiments, the modified IgG Fcregion comprises F405L and K409R mutations in the IgG Fcregion of two parental antibodies and the generation of multispecific antibody in a process known as Fab arm exchange (24).

[0189]

[0171] In some embodiments, the modified IgG Fcregion comprises Fc mutations based on the facilitate Knob-in-Hole strategy (see, e.g., Inti. Publ. No. WO 2006 / 028936). In some embodiments, an amino acid with a small side chain (hole) is introduced into one Fc domain and an amino acid with a large side chain (knob) is introduced into the other Fc domain. Without being bound by theory, co-expression of the two heavy chains leads to formation of a heterodimer as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob” (25). Exemplary Fc mutation pairs forming a knob and a hole are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V.

[0190]

[0172] In some embodiments, the modified IgG Fcregion comprises T366W and T366S / L368 A / Y407V mutations in the IgG Fcregion of two parental antibodies and the generation of multispecific antibody by co-expression or by a process known as Fab arm exchange.

[0191]

[0173] In some embodiments, modified IgG Fcregion comprises Fc mutations that facilitate the electrostatically -matched interactions strategy (26). Mutations may be engineered to generate positively charged residues at one Fc domain and negatively charged residues at the other Fc domain as described in US Patent Publ. No. US2010 / 0015133 ; US Patent Publ. No. US2009 / 0182127; US Patent Publ. No. US2010 / 028637 or US Patent Publ. No.

[0192] US2011 / 0123532. Heavy chain heterodimerization may be formed by electrostatically-matched interactions between two mutated Fc.

[0193]

[0174] In some embodiments, the antibodies disclosed herein comprise a modified Fcregion where a naturally-occurring Fc region is modified to facilitate the multimerization of the antibody upon interaction with cell surface receptors, e.g., when the antibody is a monomer in solution. The Fcmutations that facilitate antibody multimerization include, but are not limited to, E345R mutation, E430G mutation, E345R / E430G mutations, E345R / E430G / Y 440R mutations as described in (27). Such mutations may also include, but not limited to, T437R mutation, T437R / K248E mutations, T437R / K338A mutations as described in (28).

[0194]

[0175] In some embodiments, antibodies disclosed herein further comprising conservative modifications. “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur- containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis. Amino acid substitutions to the antibodies of the disclosure may be made by known methods for example by PCR mutagenesis (US Disclosure No. 4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants may be evaluated for their characteristics using assays described herein.

[0195]

[0176] In some embodiments, the antibodies disclosed herein may be post- translationally modified by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro. For example, the antibodies of the disclosure may be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation may be conducted by techniques known to those skilled in the art. In some embodiments, conjugation of antibodies with PEG enhances pharmacodynamics without disrupting antibody function.

[0196]

[0177] In some embodiments, antibodies disclosed herein are modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the disclosure. Without being bound by theory, stability of an antibody may be influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein / protein interface interactions that have impact upon the HC and LC pairing, (3) burial of polar and charged residues, (4) H- bonding network for polar and charged residues; and / or (5) surface charge and polar residue distribution among other intra- and inter-molecular forces (29). In some embodiments, potential structure destabilizing residues are identified based upon the crystal structure of the antibody or by molecular modelling in certain cases, and the effect of the residues on antibody stability are assessed by generating and evaluating variants harboring mutations in the identified residues. In some embodiments, the thermal transition midpoint (Tm), as measured by differential scanning calorimetry (DSC), is increased, indicating increased stability of the antibody. In some embodiments, the protein Tmis correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold. In some embodiments, physical stability measured (such as thermal stability) measured by DSC) correlates with physical stability measured by other methods. In some embodiments, an antibody with a higher Tmhas longer- term physical stability as compared to an antibody with a lower Tm.

[0197]

[0178] In some embodiments, antibodies disclosed herein have amino acid substitutions in the Fcregion that improve manufacturing and drug stability. In some embodiments, the amino acid substitution is H224S (or H224Q) in the hinge 221-DKTHTC-226 in IgGl (EU numbering) (“DKTHTC” disclosed as SEQ ID NO: 423) which blocks radically induced cleavage. In some embodiments, the amino acid substitution is S228P in IgG4, which blocks half-antibody exchange.

[0198] Expression and purification of antibodies

[0199]

[0179] One aspect of the present disclosure relates to production of antibodies. In some embodiments, the antibodies of the present disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences that encode the light and heavy chain polypeptides of the antibody), or by two or more separate nucleic acids, each of which encodes the light or heavy chain polypeptide of the antibody.

[0200]

[0180] In some embodiments, the nucleic acid is inserted into vectors, e.g. , nucleic acid expression vectors and / or targeting vectors. Such vectors may be used in various ways, e.g, for the expression of an antibody described herein in a cell or transgenic animal. In some embodiments, vectors are selected to be functional in the host cell in which the vector will be used. In some embodiments, a nucleic acid molecule encoding an antibody described herein is amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells. In some embodiments, the host cell is a host cell that will post-translationally modify the antibody, e.g., with glycosylation or phosphorylation. In some embodiments, the host cell will not post-translationally modify the antibody. In some embodiments, the host cell is a yeast, insect, or mammalian cell. In some embodiments, the vector is an expression vectors comprising one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0201]

[0181] In some embodiments, the antibodies disclosed herein initially comprise a leader or signal sequence at the N-terminus to guide its secretion from a host cell, but secretion of the antibodies from a host cell results in the removal of the signal peptide from the antibody. Thus, in some embodiments, the antibodies descnbed herein do not have a leader or signal sequence. In some embodiments, the antibodies comprise an altered peptidase cleavage site of a signal peptide, or pre-sequences, e.g., to increase glycosylation or yield of antibodies produced in a eukaryotic host cell expression system.

[0202]

[0182] A further aspect of the present disclosure relates to a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the disclosure. In some embodiments, the cell is any type of cell capable of being transformed with the nucleic acid or vector comprising the nucleotide sequence encoding an antibody as disclosed herein. In some embodiments, a nucleotide sequence encoding a partial and / or a full-length light and / or heavy chains are operatively linked to transcriptional and translational control sequences.

[0203]

[0183] In some embodiments, the nucleic acids and / or vectors are introduced into isolated cells (e.g., transformed), in vitro according to methods known in the art. In some embodiments, transformation of cells comprises the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA- coated microprojectiles, direct microinjection into single cells, and electroporation.

[0204]

[0184] In some embodiments, after the transformed cell is cultured under conditions suitable for expression of the encoded sequence, and the antibody, antigen binding fragment, or portion of the antibody is isolated from the cell.

[0205]

[0185] In certain embodiments, two or more vectors that together encode the antibodies described herein, are introduced into the cell.

[0206]

[0186] In some embodiments, the antibodies described herein are secreted into the cell media and purified using a technique such as affinity chromatography, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high-pressure liquid chromatography. For example, antibodies comprising an Fcregion may be purified by affinity chromatography with Protein A, which selectively binds the Fcregion.

[0207]

[0187] In some embodiments, the antibodies disclosed herein comprise affinity tags, such as hexahistidine (SEQ ID NO: 424) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl or amino terminus and purified by a one-step affinity column. For example, polyhistidine binds with great affinity and specificity to nickel, thus in some embodiments an affinity column of nickel is used for purification of polyhistidine-tagged antibodies. In some instances, more than one purification step may be employed.

[0208]

[0188] In some embodiments, a bispecific or multispecific antibody disclosed herein is generated by the process of controlled Fab arm exchange from two parental antibodies with F405L and K409R (EU numbering) mutation in IgG Fc respectively (24). Without being bound by theory, the controlled Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of Cm domains. In some embodiments, two parental antibodies are generated, one bearing the F405L Fcmutation, and one bearing the K409R Fcmutation. The heavy chain disulfide bonds in the hinge regions of the parental antibodies may be reduced and the heavy chains of the parental antibodies may be separated. In some embodiments, the F405L and K409R mutations favor heterodimerization over homodimerization of the heavy chains, e.g., so that the resulting free cysteines of one of the parental antibodies form an inter heavy -chain disulfide bond with cysteine residues of a second parental antibody, and the resulting product is a heterodimerized antibody with one half coming from one parental antibody and the other half coming from another parental antibody.

[0209]

[0189] In some embodiments, the bispecific or multispecific antibodies disclosed herein may be generated by other Fcmutations and engineering processes that facilitate Fcheterodimerization, including, but not limited to, Knob-in-Hole and the electrostatically-matched interactions.

[0210]

[0190] In some embodiments, the Knob-in-Hole strategy (see, e.g., Inti. Publ. No. WO 2006 / 028936, incorporated by reference) comprises mutating selected amino acids forming the interface of the CH3 domains in human IgG at positions affecting CH? domain interactions to promote heterodimer formation. In some embodiments, an amino acid with a small side chain (hole) is introduced into one Fcdomain and an amino acid with a large side chain (knob) is introduced into the other Fcdomain of the parental antibodies, and after co-expression of the two heavy chains, a heterodimer is formed, e.g., because of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob.” Exemplary Cm substitution pairs forming a knob and a hole include: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A / Y407V.

[0211]

[0191] In some embodiments, the electrostatically-matched interactions strategy is used to generate positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface as described in US 2010 / 0015133 Al; US 2009 / 0182127 Al; US 2010 / 028637 Al, or US 2011 / 0123532 Al. In some embodiments, heterodimerization of heavy chains can be formed by electrostatically-matched interactions between two mutated Fc.

[0212]

[0192] In some embodiments, the formation of bispecific or multispecific antibody is assessed, e.g., by analytical HPLC, if there is a detectable difference in the biophysical properties of the two parental antibodies. In some embodiments, a difference in pl may lead to two separate peaks for the two parental antibodies on Cation Exchange chromatography and the bispecific antibody may migrate as a peak in between. In some embodiments, a difference in hydrophobicity may lead to two separate peaks for the two parental antibodies on hydrophobic interaction chromatography and the bispecific antibody may migrate as a peak in between. In some embodiments, analytical HPLC is used to demonstrate the formation of bispecific antibody and / or quantitate the percentage of bispecific or multispecific antibody formed.

[0213] Anti-CD318 antibody-based antibody conjugate

[0214]

[0193] Another aspect of the present disclosure relates to an antibody conjugate comprising an antibody moiety conjugated to a payload, wherein the antibody moiety is the anti- CD318 antibody or antigen-binding fragment as disclosed herein, the anti-CD318 based immunocytokine as disclosed herein, or the multispecific antibody or antigen-binding fragment as disclosed herein. In some embodiments, the antibody conjugate comprises anti-CD318 antibody or antigen-binding fragment and a payload. In some embodiments, the payload is a drug payload or a diagnostic agent. In some embodiments, the payload is a cytotoxic drug. In some embodiments, the antibody moiety is conjugated to the payload through a cleavable or non- cleavable chemical linker.

[0215]

[0194] In some embodiments, the payload is a cytotoxin, a radioisotope, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a hormone, hormonal antagonist, enzyme, protein degrader, oligonucleotide, DMA, RNA, siRNA, shRNA, microRNA. mRNA, photoactive therapeutic agent, anti -angiogenic agent, pro-apoptotic agent, peptide, lipid, hydrate carbon, a chelating agent, or any combination thereof

[0216]

[0195] In some embodiments, the present disclosure provides an anti-CD318 antibodybased antibody drug conjugate (CD318-ADC) by conjugation of cytotoxic drug payload onto the anti-CD318 antibodies disclosed herein through a chemical linker. In some embodiments, anti- CD318 antibody binding to cell surface CD318 antigens leads to internalization of the CD318- ADC whereupon the cytotoxic drug component is released and weakens or kills CD318- expressing tumor cells.

[0217]

[0196] In some embodiments, the anti-CD318 antibody-based ADC comprises an antibody targeting CD318 (e.g., humanized antibody 9A2), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 7, 8, and 9, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 10, 11, and 12, respectively. In some embodiment, the antibody targeting CD318 (e.g., humanized antibody 9A2) comprises a heavy chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 101 or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 101, and / or a light chain variable region comprising an amino acid selected from SEQ ID NOs: 102 and 103, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 102 and 103.

[0218]

[0197] In some embodiments, the anti-CD318 antibody-based ADC comprises an antibody targeting CD318 (e.g., humanized antibody 3A11), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 13, 14, and 15, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 16, 17, and 18, respectively. In some embodiment, the antibody targeting CD318 (e.g., 3A11) comprises a heavy chain variable region comprising an amino acid sequence as set forth as SEQ ID NO: 104 or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 104, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 105, 106 and 107, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 105, 106 and 107.

[0219]

[0198] In some embodiments, the anti-CD318 antibody-based ADC comprises an antibody targeting CD318 (e.g., humanized antibody 6662), wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 19, 20, and 21, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 22, 23, and 24, respectively. In some embodiment, the antibody targeting CD318 (e.g., 6662) comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 108 and 109, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 108 and 109, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 110 and 111, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 110 and 111.

[0220]

[0199] In some embodiments, the anti-CD318 antibody-based ADC comprises the anti- CD318 antibody 9A2, wherein the anti-CD318 antibody comprises an anti-CD318 antibody heavy chain variable region and an IgGl Fc selected from SEQ ID NOs: 201 and 212, or comprises an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 201 and 212, and an anti-CD318 antibody light chain comprising an amino acid sequence selected from SEQ ID NOs: 202 and 203, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 202 and 203.

[0221]

[0200] In some embodiments, the anti-CD318 antibody-based ADC comprises the anti- CD318 antibody 3A11, wherein the anti-CD318 antibody comprises an anti-CD318 antibody heavy chain variable region and a IgGl Fc selected from SEQ ID NOs: 204 and 213, or comprises an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 204 and 213, and an anti-CD318 antibody light chain comprising an amino acid sequence selected from SEQ ID NOs: 205, 206 and 207, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 205, 206 and 207.

[0222]

[0201] In some embodiments, the anti-CD318 antibody-based ADC comprises the anti- CD318 antibody 6662, wherein the anti-CD318 antibody comprises an anti-CD318 antibody heavy chain variable region and a IgGl Fc selected from SEQ ID NOs: 208, 209 and 214, or comprising an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 208, 209 and 214, and an anti-CD318 antibody light chain comprising an amino acid sequence chosen from SEQ ID NOs: 210 and 211, or comprising an ammo acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99%) identity to any one of SEQ ID NOs: 210 and 211.

[0223]

[0202] In some embodiments, the anti-CD318 antibody-based ADC comprises a bispecific or multispecific antibody comprising the anti-CD318 antibodies disclosed herein and antibodies recognizing other tumor antigens. In some embodiments, bispecific or multispecific antibody-based ADC recognizes multiple antigens and eliminates tumor cells expressing these different antigens including CD318.

[0224]

[0203] In some embodiments, the anti-CD318 antibody-based ADC comprises the anti- CD318 antibodies disclosed herein conjugated to a payload, e.g., through cleavable and / or non- cleavable chemical linkers. In some embodiments, the drug payload has cytotoxicity to the CD318-expressing cells.

[0204] In some embodiments, the payload is a cytotoxic drug payload. In some embodiments, the anti-CD318 antibody-based ADC comprises a tubulin inhibitor. In some embodiments, the tubulin inhibitor blocks tubulin polymerization, e.g., acting as an anti-mitotic agent, leading to cell cycle arrest and apoptosis. As non-limiting examples, the tubulin inhibitor type of cytotoxic drug payloads include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1 (N2'-deacetyl-N2'- (3-mercapto-l-oxopropyl)-maytansine, DM4 (N2'-deacetyl-N2'- (4-mercapto-4-methyl-l-oxopentyl)-maytansine), and tubulysin.

[0225]

[0205] In some embodiments, the cytotoxic drug payload comprises a topoisomerase inhibitor. The topoisomerase inhibitor may disrupt DNA replication and transcription that leads to cell cytotoxicity. As non-limiting examples, the topoisomerase inhibitor type of cytotoxic drug payload include DXd (N-[(lS,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4- methyl- 10,13-dioxo- 1H, 12H-benzo[de]pyrano[3',4' : 6,7]indolizino[ 1 ,2-b] quinolin- 1 -yl] -2- hydroxyacetamide), SN-38 ((S)-4,l l-diethyl-4-hydroxy-9-hydroxymethyl-lH- pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3,14(4H,12H)-dione-5, 10-dione), PNU-159682, and doxorubicin ((8S,10S)-10-[(2S,4S,5S,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-8- glycosyl-7,8,9,10-tetrahydro-6,8,l l-trihydroxy-l-methoxy-5,12-naphthacenedione).

[0226]

[0206] In some embodiments, cytotoxic drug payload comprises a DNA-damaging agent. The DNA-damaging agent may cause breaks or cross-links in DNA that leads to cell cytotoxicity. As non-limiting examples, the DNA-damaging agent type of cytotoxic drug payload include duocarmycin (N-[(lS,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9- hydroxy-4-methyl-10,13-dioxo-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l- yl]-2-hydroxy acetamide), calicheamicin, pyrrolobenzodiazepines (PBDs).

[0227]

[0207] In some embodiments, the cytotoxic drug payload comprises a RNA polymerase II inhibitors. The RNA polymerase II inhibitor may inhibit the synthesis messenger RNA (mRNA) that leads to cell cytotoxicity. As non-limiting examples, the RNA polymerase II inhibitor type of cytotoxic drug payload include alpha-amanitin ((!S,3R,4aS,9S,9aR,10aS)-9- ethyl-l-methyl-lH-pyrano[4',3':4,5]oxazolo[2,3-c][l,4]oxazine-3,14(4H,12H)-dione).

[0228]

[0208] In some embodiments, the cytotoxic drug payload comprises a protein synthesis inhibitor. The protein synthesis inhibitor may inhibit protein synthesis that leads to cell cytotoxicity. As non-limiting examples, the protein synthesis inhibitor type of cytotoxic drug payload include auristatins and maytansinoids.

[0229]

[0209] In some embodiments, the cytotoxic drug payload comprises a protein-degrader- recruiting molecule. The protein-degrader-recruiting molecule may facilitate the degradation of proteins key to cell survival and proliferation by ubiquitin-proteasome system that leads to cell cytotoxicity. As non-limiting examples, the protein-degrader-recruiting molecule type of cytotoxic drug payload include proteolysis targeting chimaeras (PROTACs) and molecular glues.

[0230]

[0210] In some embodiments, the drug payload (e.g., cytotoxic drug pay load) comprises the same drug payload or same type of drug payload conjugated on every single anti- CD318 antibody molecule. In some embodiments, the cytotoxic drug payload comprises a different drug payload or different type of drug payload conjugated on each single anti-CD318 antibody molecule.

[0231]

[0211] In some embodiments, the drug pay load (e.g., cytotoxic drug pay load) is conjugated to the anti-CD318 antibody through one or more chemical linkers. In some embodiments, chemical linkers enable the cytotoxic payloads to remain attached to the antibody until the target is reached. In some embodiments, the linkers include non-cleavable linkers which are composed of stable bonds that resist proteolytic degradation and cleavable linkers that are degraded by tumor-associated factors (such as the acidic and / or reducing conditions associated with most tumors or intracellular proteases) and enable the efficient release of active payloads upon internalization into cancer cells.

[0232]

[0212] In some embodiments, the drug pay load (e.g., cytotoxic drug pay load) is conjugated to the anti-CD318 antibody through a non-cleavable linker. In some embodiments, the non-cleavable linker is stable and releases the payload only after lysosomal degradation of the ADC molecule once internalized in the cells. As non-limiting examples, the non-cleavable linkers include maleimidocaproyl (MC) and SMCC (N-succinimidyl 4- (maleimidomethyl)cyclohexane-l-carboxylate).

[0233]

[0213] In some embodiments, the drug payload (e.g., cytotoxic drug payload) is conjugated to the anti-CD318 antibody through a disulfide ty pe of cleavable linkers. In some embodiments, the disulfide linker is cleaved in the presence of reducing agents like glutathione, which are abundant inside cells. As non-limiting examples, the disulfide linkers include SPDB (N-succinimidyl 4-(2-pyridyldithio)butanoate).

[0234]

[0214] In some embodiments, the drug pay load (e.g., cytotoxic drug pay load) is conjugated to the anti-CD318 antibody through a peptide type of cleavable linker. In some embodiments, the peptide linker is cleaved by specific proteases (e.g., cathepsin B) found in the lysosomes. As non-limiting examples, the peptide linkers include Valine-Citrulline (Val-Cit).

[0235]

[0215] In some embodiments, the drug payload (e.g., cytotoxic drug payload) is conjugated to the anti-CD318 antibody through a hydrazone type of cleavable linker. In some embodiments, the hydrazone linker is cleaved in the acidic environment of the lysosome or endosome. As non-limiting examples, the hydrazone linkers include 4-(4'-Acetoxyphenyl)-2- butanone Hydrazone.

[0216] In some embodiments, drug payload (e.g., cytotoxic drug payload) is conjugated to the anti-CD318 antibody through a carbonate type of cleavable linker. In some embodiments, the carbonate linker is cleaved in the acidic environment of the lysosome or endosome. As nonlimiting examples, the carbonate linkers include 4-(4'-Acetoxyphenyl)-2-butanone Carbonate.

[0236]

[0217] In some embodiments, the drug pay load (e.g., cytotoxic drug pay load) is conjugated to the anti-CD318 antibody through a glycosidic type of cleavable linker. In some embodiments, the glycosidic linker is cleaved by (3-glucuromdase present in lysosomes.

[0237]

[0218] In some embodiments, the drug pay load (e.g., cytotoxic drug pay load) is conjugated to the anti-CD318 antibody through cysteine conjugation. In some embodiments, the process involves the reduction of disulfide bonds in the antibody to expose free thiol groups followed by the employment of maleimide reagents that form stable thioester bonds with cysteine’s thiol groups to conjugate drug payload to the antibody. In some embodiments, different drug-to-antibody ratios (DARs) are achieved, e.g., through controlled conjugation reactions.

[0238]

[0219] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of one (one drug payload conjugated per antibody molecule).

[0239]

[0220] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of two (two drug payloads conjugated per antibody molecule).

[0240]

[0221] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of three (three drug payloads conjugated per antibody molecule).

[0241]

[0222] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of four (four drug payloads conjugated per antibody molecule).

[0242]

[0223] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of five (five drug pay loads conjugated per antibody molecule).

[0243]

[0224] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of six (six drug payloads conjugated per antibody molecule).

[0244]

[0225] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of seven (seven drug payloads conjugated per antibody molecule).

[0245]

[0226] In some embodiments, the anti-CD318 antibody-based ADC has a DAR of eight (eight drug payloads conjugated per antibody molecule).

[0246]

[0227] In some embodiments, the anti-CD318 antibody-based ADC is a homogenous population of a single anti-CD318 antibody-based ADC molecule with a defined DAR or a heterogenous population of a mixture of anti-CD318 antibody-based ADC molecules with different DAR numbers.

[0228] In some embodiments, the anti-CD318 antibody-based ADC is generated through lysine conjugation. In some embodiments, chemical reagents such as NHS esters react with the lysine’s primary amines, leading to the attachment of drug payload to the amino groups present on the lysine residues of the antibody.

[0247]

[0229] In some embodiments, the anti-CD318 antibody-based ADC is generated through site-specific conjugation. In some embodiments, specific sites on the antibody, such as Fc domain, engineered cysteine residues or glycosylation sites, are targeted to achieve a uniform DAR. As non-limiting examples, site-specific conjugation includes Light-Activated Site-Specific Conjugation (LASIC) with oYo-link reagents, THIOMAB™ antibodies and glycosyl remodeling.

[0248]

[0230] In some embodiments, the anti-CD318 antibody-based ADC is generated through click chemistry. In some embodiments, bioorthogonal reactions, such as copper- catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC), are used to attach the drug payload to the antibody.

[0249]

[0231] In some embodiments, the anti-CD318 antibody-based ADC is generated through transglutaminase-mediated conjugation. In some embodiments, the enzyme transglutaminase is used to form covalent bonds between glutamine residues on the antibody and lysine residues on the drug payload.

[0250]

[0232] In some embodiments, the drug-antibody ratio (DAR) and homogeneity of anti- CD318 antibody -based ADC is assessed by High-Performance Liquid Chromatography (HPLC), e.g., Reversed-phase HPLC (RP-HPLC) and hydrophobic Interaction Chromatography (HIC). In some embodiments, information on the molecular weight and structure of the ADC is evaluated by mass spectrometry such as Electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In some embodiments, size-Exclusion Chromatography (SEC) is used to separate ADCs based on their size and to assess aggregation.

[0251] Target binding and functional activity of anti-CD318 antibody-based molecules

[0252]

[0233] An aspect of the present disclosure relates to determining the binding and functional activity of anti-CD318 antibodies and / or anti-CD318 antibody-based molecules. In vitro binding and cell-based assays known in the art may be used to in determining the anti- CD318 antibodies and / or anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein in binding to their target antigens. For example, the binding of antibodies disclosed herein may be determined by ELISA by immobilizing recombinant or purified antigen, sequestering the antibody with the immobilized antigen and determining the amount of bound antibody. In some embodiments, binding is determined by Surface Plasmon Resonance (SPR) or Bio-layer interferometry (BLI) for kinetic analysis of binding interactions. In some embodiments, cell-based binding assays comprise determining the binding of antibody by incubating the antibody with cells expressing antigens on cell surface and determining the amount of antibody bound to cell surface antigen, e.g., by flow cytometry.

[0253]

[0234] In vitro and cell-based functional assays known in the art may be used in determining the functional activity of the anti-CD318 antibody-based immunocytokines disclosed herein. In some embodiments, the functional activities of the IFNa components of the anti-CD318 antibody based immunocytokines with IFNa, as described herein, are assessed by reporter-based cell assay for IFNa. In some embodiments, the effects on the activation and proliferation of immune cells by the anti-CD318 antibody -based immunocytokines of the present disclosure are assessed by mixing the immunocytokines with purified T cells or PBMC followed by the quantitation of activation and proliferation markers of specific immune cells. In some embodiments, the cytotoxicity of tumor cells by the anti-CD318 antibody -based immunocytokines of the present disclosure are assessed by mixing the immunocytokines with tumor target cells followed by the quantitation of remaining viable cells by a cell cytotoxicity assay kit.

[0254]

[0235] In vitro and cell-based functional assays known in the art may be used in determining the functional activity of the anti-CD318 antibody-based CD318 x VEGF x PD-L1 multispecific antibodies disclosed herein. In some embodiments, the functional activities of anti- VEGF and anti-PD-Ll components of the CD318 x VEGF x PD-L1 multispecific antibodies described herein are assessed by reporter-based cell assays. In some embodiments, the effects of CD318 x VEGF x PD-L1 multispecific antibodies in the sequestration of free VEGF secreted by target cells are assessed by incubating the multispecific antibody with target cells followed by the quantitation of free VEGF by an ELISA kit. In some embodiments, the effects of CD318 x VEGF x PD-L1 multispecific antibodies in the neutralization of VEGF activity are also be assessed by incubating the multispecific antibody with human umbilical vein endothelial cells (HUVEC) followed by the quantitation of the proliferation and migration of HUVEC cells. In some embodiments, the effects of CD318 x VEGF x PD-L1 multispecific antibodies on the stimulation of the activation, proliferation and functionality of T cells are assessed by T cell activation and proliferation assays and T cell mediated cytotoxicity assays with purified T cells or PBMC. In some embodiments, the cytotoxicity of tumor cells by the CD318 x VEGF x PD-L1 multispecific antibodies of the present disclosure is assessed by mixing the test articles with tumor target cells and PBMC followed by the quantitation of remaining viable target cells by a cell cytotoxicity assay kit.

[0236] In vitro and cell-based functional assays known in the art may be used in determining the functional activity of the anti-CD318 antibody-based drug conjugates disclosed herein. In some embodiments, the internalization of the anti-CD318 antibody based ADC described herein is assessed by fluorescence based cell internalization assays. In some embodiments, the cytotoxicity of tumor cells by the anti-CD318 antibody -based ADC of the present disclosure is assessed by mixing the test articles with tumor target cells followed by the quantitation of remaining viable cells by a cell cytotoxicity assay kit.

[0255]

[0237] In addition to in vitro functional assays, animal models and ex vivo functional assays known in the art may be used in determining the in vivo efficacy of the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein. In some embodiments, the anti-tumor efficacy of the molecules described herein is assessed by dosing mice xenografted with tumors derived from human tumor cell lines and patient derived primary tumor cells followed by the measurement of tumor size.

[0256] Pharmaceutical Compositions

[0257]

[0238] A further aspect of the present disclosure relates to a pharmaceutical composition comprising the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, or the antibody conjugate as disclosed herein, and a pharmaceutically acceptable agent described herein. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one of the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein, e.g., for use in the methods herein. In some embodiments, the pharmaceutical composition comprises a therapeutically or prophylactically effective amount of the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates described herein, mixed with a suitable earner, e.g., a pharmaceutically acceptable agent. In some embodiments, the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates described in this disclosure are sufficiently purified for administration to an animal before formulation in a pharmaceutical composition.

[0258]

[0239] Pharmaceutically acceptable agents may include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

[0240] In some embodiments, the composition is in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0259]

[0241] In some embodiments, the composition is suitable for parenteral administration. Exempt ary compositions are suitable for injection or infusion into an animal by any route available to a skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebro ventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral, or inhaled routes.

[0260]

[0242] In some embodiments, pharmaceutical compositions described herein are formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) for a sustained release and / or increased stability or half-life in a particular local environment.

[0261] Methods of Use

[0262]

[0243] An aspect of the present disclosure relates to the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein for the treatment or prevention of cancers, such as breast, bladder, colon, lung, pancreatic, prostate and other cancer. In some embodiments, the cancer is selected from breast, bladder, colon, lung, pancreatic, prostate, and any cancer with TAA (e.g., CD318) expression. In some embodiments, cancer is any cancer with CD318 expression.

[0263]

[0244] Another aspect of the present disclosure relates to the use of anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein or a composition comprising anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein in the manufacture of a medicament for treating or preventing a disease or condition, e.g., cancer, such as breast, bladder, colon, lung, pancreatic, prostate and other cancer. In some embodiments, the cancer is selected from breast, bladder, colon, lung, pancreatic, prostate, and any cancer with TAA (e.g., CD318) expression. In some embodiments, cancer is any cancer with CD318 expression.

[0264]

[0245] A further aspect of the present disclosure relates to anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein or a composition comprising the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein for use in a method of treating or preventing a disease or condition, e.g., cancer, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the anti-CD318 antibody-based immunocytokines, multispecific antibodies and antibody drug conjugates or the composition. In some embodiments, the cancer is selected from breast, bladder, colon, lung, pancreatic, prostate, and any cancer with TAA (e.g., CD318) expression. In some embodiments, cancer is any cancer with CD318 expression.

[0265]

[0246] Another aspect of the present disclosure relates to anti-CD318 antibody -based immunocytokines, multispecific antibodies and antibody drug conjugates disclosed herein for use in a combination regimen, e.g., with chemotherapy, radiotherapy, and / or cell therapy.

[0266]

[0247] A further aspect of the present disclosure relates to a method for treating or preventing a disease or condition associated with overexpression of CD318 in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, the antibody conjugate as disclosed herein, or the pharmaceutical composition as disclosed herein.

[0267]

[0248] A further aspect of the present disclosure relates to a method for inhibiting expression of CD318 in a cell, comprising contacting the cell with the anti-CD318 antibody or antigen-binding fragment as disclosed herein, the immunocytokine as disclosed herein, the multispecific antibody or antigen-binding fragment as disclosed herein, the antibody conjugate as disclosed herein, or the pharmaceutical composition as disclosed herein.

[0268]

[0249] All combinations of the various elements described herein are within the scope of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0269]

[0250] This disclosure will be better understood from the following Experimental Details. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the disclosure.

[0270] EXAMPLES

[0271] Example 1: CD318 binding by the three mouse anti-human CD318 antibodies

[0272]

[0251] Three monoclonal mouse anti -human CD318 antibodies with clone numbers 9A2, 3 Al 1, and 6662 respectively were characterized for their binding to human and mouse CD318 recombinant proteins by ELISA assays. In this assay, 1 pg / mL human and mouse full length CD318 as fusion proteins with human IgG Fc were coated on ELISA plate. Increasing concentrations of CD318-9A2, CD318-3A11, and CD318-6662 antibodies were applied on the CD318 antigen-coated ELISA plate and their binding to the recombinant human and mouse CD318 antigens were detected by HRP-conjugated anti-mouse IgG secondary antibody. All three anti-CD318 antibodies concentration-dependently bound to recombinant human CD318 protein (Figure 1A). However, only CD318-9A2 antibody concentration-dependently bound to mouse CD318 protein while CD318-3 All or CD318-6662 antibody did not bind to mouse CD318 (Figure IB). Besides the full length human CD318, the binding of these three antibodies to a truncated isoform of human CD318 with amino acids 30 to 343 containing the N-terminal CUB1 domain were also evaluated by ELISA binding assays. All three antibodies concentration- dependently bound to this truncated CD318 protein (Figure 2A, 2B).

[0273]

[0252] Besides binding to recombinant CD318 antigen, flow cy lomelry-based binding assays were employed to evaluate the binding to CD318 expressed on surface of tumor cells. In this assay, increasing concentrations of mouse anti -human CD318 antibodies were applied to tumor cells and their bindings to cell surface CD318 were detected by PE-conjugated secondary' antibody by MacsQuant flow cytometer. All three anti-CD318 antibodies concentration- dependently bound to CD318 expressed on breast cancer cell line T-47D (Figure 3 A) and pancreatic cell line BxPC-3 (Figure 3B). The CD318-3 Al l and CD318-6662 antibodies showed better potencies and efficacies in CD318 binding relative to CD318-9A2 antibody.

[0274] Example 2: Humanization of the three mouse anti-human CD318 antibodies

[0275]

[0253] The three mouse anti-human CD318 antibodies were sequenced by enzyme digestions followed by Liquid Chromatography with tandem mass spectrometry LC-MS / MS. The mouse anti-CD318 9A2 antibody comprises a heavy chain variable region sequence set forth as SEQ ID NO: 1 and a light chain variable region sequence set forth as SEQ ID NO: 2. The mouse anti-CD318 3 Al l antibody comprises a heavy chain variable region sequence set forth as SEQ ID NO. 3 and a light chain variable region sequence set forth as SEQ ID NO: 4. The mouse anti-CD318 6662 antibody comprises a heavy chain variable region sequence set forth as SEQ ID NO. 5 and a light chain variable region sequence set forth as SEQ ID NO: 6.

[0276]

[0254] The three monoclonal mouse anti -human CD318 antibodies were humanized with the inclusion of mouse CDRs into human germline scaffolds while preserving a few key framework residues to achieve higher stability, better recombinant protein expression, and minimization of immunogenicity. Plasmids encoding combinations of humanized heavy chain variants and humanized light chain variants listed in Table 2 were co-transfected into Expi293F cells following the transfection kit instructions (Thermo Scientific) to express humanized anti- CD318 antibodies. Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 pm filter. The purifications of expressed antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm. All the humanized anti-CD318 antibodies except anti-CD318-3 Al l VHhumlVLhum3_IgGl were successfully expressed and purified. Table 2. Chain Assembly of Humanized anti-CD318 Antibodies

[0277]

[0255] ELISA-based binding assays were employed to evaluate the binding to human CD318 by the humanized antibodies. In this assay, 1 pg / mL human CD318 as fusion proteins with human IgG Fc were coated on ELISA plate. Increasing concentrations of humanized anti- CD318 antibodies were applied on the coated ELISA plate and their binding to the recombinant CD318 were detected by HRP -conjugated anti-human IgG F(ab)’ secondary antibody. All the tested humanized antibodies concentration-dependently bound to recombinant CD318 with compatible potencies to the corresponding mouse anti-CD318 antibodies 9A2, 3A11, and 6662 clones (Figure 4A-4C). Besides, the humanized 9A2 antibodies concentration-dependently bound to mouse CD318 as the corresponding mouse 9A2 antibody while neither humanized

[0278] 3 Al l antibodies nor humanized 6662 antibodies bound to mouse CD318 as their mouse antibody counterparts.

[0279]

[0256] Flow cytometry-based binding assays were also employed to evaluate the binding to human CD318 expressed on T-47D cells by humanized CD318 antibodies. In this assay, increasing concentrations of humanized CD318 antibodies were applied to T-47D cells, and their bindings to cell surface CD318 were detected by PE-conjugated anti -human secondary antibody by MacsQuant flow cytometer. All the tested humanized antibodies concentration- dependently bound to cell surface CD318 with compatible potencies to the corresponding mouse anti-CD318 antibodies 9A2, 3A11 and 6662 clones (Figure 5A-5C).

[0280] Example 3: Expression of CD318 in tumor cell lines

[0281]

[0257] The expression of CD318 in cell lines derived from different types of human tumors were studied by flow cytometry assay. In this assay, 1 pg / mL CD318-3A11 VhhumlVlhum2_IgGl antibody was applied to tumor cells and its binding to cell surface CD318 was detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. In some cases, an anti-HER2 antibody was also included in the assay to quantitate HER2 expression in the tumor cells lines in order to compare the expression levels of CD318 and HER2. It was observed that CD318 had relatively high expression levels in exemplary cell lines derived from breast cancer (Figure 6A), pancreatic cancer (Figure 6B), lung cancer (Figure 6C), esophagus cancer (Figure 6D), gastric cancer (Figure 6E) and colon cancer (Figure 6F). In most cases, the levels of CD318 expression were relatively higher than that of HER2 expression.

[0282] Example 4: Cytotoxicity of tumor cells by CD318 antibodies fusion with IFNa

[0283]

[0258] To explore whether CD318 can be employed as a tumor associated antigen to mediate the cytotoxicity of tumor cells, an immunocytokine approach was attempted to assess whether CD318 antibodies fused with IFNa can kill the tumor cells. CD318 antibody heavy chains were designed with native human IFNa2b and attenuated IFNa2b with LI 5 A mutation (h IFNa2b_L15A), L15A and L153K mutations (h IFNa2b_L15A_L153K), or L30A mutation (h IFNa2b_L30A) fused at the C-terminus of exemplary humanized CD318-9A2, CD318-3A11 and CD318-6662 antibody heavy chains. These heavy chains with IFNa2b fusions paired with corresponding light chains were expressed in Expi293 cells to make exemplary immunocytokines with CD318 antibody fused with IFNa2b listed in Table 3.

[0284] Table 3. Humanized anti-CD318 antibody fusion with human IFNa2b

[0259] Functional activities of the exemplary CD318 antibody fusions with hTFNo.2b in activating type 1 IFN receptors were assessed by HEK-Blue IFNa / p reporter assay (Invivogen). In this assay, increasing amounts of the CD318 antibody fusions with hIFNcc2b were incubated with reporter cells and the hIFNa2b-driven expression of SEAP reporter gene was quantitated. All CD318 antibody fusions with hIFNa2b concentration-dependently activated reporter gene expression (Figure 7A to 7C). The native hIFNa2b mediated the most potent reporter gene expression, while the hIFNa2b with L15A mutation, L15A and L153K mutations and L30A mutation mediated increasingly attenuated levels of reporter gene expression.

[0285]

[0260] Besides the reporter assays, the cytotoxicity of CD318-expressing tumor cells by CD318 antibody fusions with hIFNa2b were assessed by cell proliferation assays. In these assays, increasing amounts of CD318 antibody 9A2 and 3A11 fusions with hIFNcc2b were incubated with lung cancer cell line HCC827 and pancreatic cancer cell line BxPC-3. The hIFN«2b-driven cytotoxicity of CD318 expressing tumor cells were assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega). All CD318 antibody fusions with hIFNa2b could concentration-dependently inhibit the proliferation of HCC827 cells (Figure 8A to 8B) and BxPC-3 cells (Figure 8C to 8D). The CD318-mediated effects were confirmed by the significant differences in the cell cytotoxicity potency mediated by hIFNa2b fusion with anti-CD318 antibody when compared to the null control anti-gp!20 antibody. The native hIFNa2b mediated the most potent cell cytotoxicity while the hIFNa2b with L15A mutation, L15A and L153K mutations, and L30A mutation mediated increasingly attenuated potencies in cell cytotoxicity.

[0286]

[0261] Besides direct cytotoxicity in CD318-expressing tumor cells, the capability of hIFNa2b in stimulating immune effector cells from PBMC, including T cells and NK cells, to kill the tumor cells was also assessed by using the PBMC-mediated cell cytotoxicity assay. In this assay, PBMCs were pre-incubated with 100 pM CD318 antibody 9A2 or CD318 antibody 9A2 fusions with native or attenuated hIFNa2b. Forty-eight hours later, pre-treated antibodies were washed, and new antibodies were added at the concentration of 10 nM. PBMCs were incubated with pancreatic cancer cell BxPC-3 for another four hours with the effective to target cell ratio from 20: 1 to 0.625: 1. PBMC and the hIFNa2b-driven cytotoxicity of fluorescence- labeled BxPC-3 cells were assessed by flow cytometry-based assay. It was observed that CD318 antibody fusions with hIFNa2b concentration-dependently mediated the lysis of BxPC-3 cells while neither isotype control IgG antibody nor the CD318-9A2 antibody without hIFNa2b fusion showed cell cytotoxicity activities (Figure 9). The native hIFNa2b mediated the most potent level of cell cytotoxicity while the hIFNa2b with L15A mutation, L15A and L153K mutations, and L30A mutation mediated increasingly attenuated potencies of cell cytotoxicity.

[0262] The efficacy of CD318 antibody fusions with hIFNa2b to inhibit the in vivo growth of the BxPC-3 cells was evaluated in a tumor xenograft model. In this model, the CD318-expressing BxPC-3 cells were subcutaneously inoculated into NCG mice to form a tumor with 5 mice per study group. Human donor PBMC were intravenously injected into the mice 5-7 days before tumor cell grafting. CD318 9A2 antibody fusions with hIFNa2b were dosed intraperitoneally at 1 mg / kg twice per week starting at day 0 post tumor cell inoculation for three weeks. The volume of the tumor and the body weight of mice were measured. The CD318 9A2 antibody fusion with hIFNoc2b_L15A was observed to have a 50% BxPC-3 tumor cell growth inhibition compared to the PBS treatment group. The more severely attenuated hIFN«2b (hIFNa2b_L15A_L153K and hIFNa2b_L30A) as CD318-9A2 fusion showed insignificant levels of tumor growth inhibition at this dosing level (Figure 10A). There were minimal differences in body weight between antibody treatment group and vehicle control group (Figure 10B). This data demonstrated the tumor killing efficacy of CD318 antibody fusions with hIFNa2b_L15A.

[0287] Example 5: Cytotoxicity of tumor cells by a multispecific antibody targeting CD318, VEGF and PD-L1

[0288]

[0263] To explore whether CD318 can be employed as a tumor associated antigen to mediate the cytotoxicity of tumor cells, a multispecific antibody targeting CD318, VEGF and PD-L1 was developed. Mediated by CD318 antibody binding to cell surface antigen, the multispecific antibody can guide VEGF neutralization activity around CD318-expressing cells to block angiogenesis and also to target PD1-PD-L1 blockade activity to stimulate T cells to kill CD318-expressing cells. The CD318 x VEGF x PD-L1 multispecific antibody comprises a CD318 antibody Fab as the CD318-binding arm and a VEGF antibody Fab as the VEGF binding arm, an IgGl Fc with an anti-PD-Ll antibody Fab in a single chain Fv (ScFv) format fused at the C -terminus of both heavy chains. To generate an exemplary multispecific antibody CD318-9A2 x VEGF x PDLlScFv, two parental antibodies for each binding arm were made followed by heterodimerization of the two parental antibodies through a controlled Fab-arm exchange process. One parental antibody comprises humanized CD318-9A2 antibody heavy chain 9A2 VHhuml with an IgGl Fc with F405L mutation and a PD-L1 antibody Fab as ScFv fused at the C -terminus as set forth as SEQ ID No. 401 and the humanized CD318-9A2 antibody light chain 9A2 VLhuml as set forth as SEQ ID No. 202. Another parental antibody comprises an anti- VEGF antibody heavy chain with an IgGl Fc with K409R mutation and a PD-L1 antibody Fab in a ScFv format fused at the C-terminus as set forth as SEQ ID No. 402 and an anti-VEGF antibody light chain as set forth as SEQ ID No. 403. Plasmids encoding heavy chain and light chain of each parental antibody were co-transfected into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 pm filter. The expressed parental antibodies were purified by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified parental antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm. For controlled Fab-arm exchange, equal molar amounts of both parental antibodies were mixed and reduced for 5 hours in the presence of 75 mM 2-mercaptoethylamine (2-MEA). The reaction mixture was dialyzed against DPBS to allow the multispecific antibody CD318- 9A2VHhumlVLhuml x VEGF_IgGl_PDL1996-ScFv (abbreviated as CD318-9A2 x VEGF x PDLlScFv) formation.

[0289]

[0264] Flow cytometry-based binding assay was employed to evaluate the binding to human CD318 expressed on BxPC-3 cells by CD318-9A2 x VEGF x PDLlScFv multispecific antibody and CD318-9A2 humanized antibody as a comparison. In this assay, increasing concentrations of antibodies were applied to BxPC-3 cells and their bindings to cell surface CD318 were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. All the tested antibodies concentration-dependently bound to cell surface CD318 (Figure 11A). However, the CD318-9A2 x VEGF x PDLlScFv multispecific antibody was 20- fold less potent than CD318-9A2 in binding CD318 due to the presence of only one CD318 binding arm on the multispecific antibody.

[0290]

[0265] A VEGF reporter assay (Promega) was adopted to assess the blockade of VEGF signalling by CD318-9A2 x VEGF x PDLlScFv multispecific antibody and anti-VEGF antibody as a comparison. In this assay, increasing concentrations of testing antibodies were incubated with 25 ng / mL VEGF along with the VEGF reporter cell line. The blockade of luciferase reporter gene was quantitated by Bio-Gio luminescence kit (Promega). It was observed that both testing antibodies concentration-dependently blocked VEGF signaling (Figure 1 IB). The blockade by the CD318-9A2 x VEGF x PDLlScFv multispecific antibody was just slightly weaker in potency than the anti-VEGF antibody although there is only one VEGF-binding arm on the multispecific antibody.

[0291]

[0266] The functional blockade of PD-L1 / PD-1 interaction by CD318-9A2 x VEGF x PDLlScFv multispecific antibody was evaluated in a PD-L1 blockade reporter assay (Invivogen). Increasing concentrations of CD318-9A2 x VEGF x PDLlScFv multispecific antibody and PD-L1 antibody atezolizumab were applied to Jurkat cell-based effector reporter cells with the expression of T cell receptor (TCR) and PD-1, and Raji cell-based antigen presentation cell (APC) with the expression of PD-L1 (Invivogen). The blockade of PD-1 / PD-L1 interaction was quantitated via stimulated luciferase reporter gene expression. The tested antibodies showed concentration-dependent reporter gene expression activation (Figure 11C). The CD318-9A2 x VEGF x PDLlScFv multispecific antibody with two PD-L1 ScFv domains showed about 8-fold less potency in blocking PD-L1 activity relative to atezolizumab.

[0292]

[0267] The efficacy of CD318-9A2 x VEGF x PDLlScFv multispecific antibody in the inhibition of CD318-expressing tumor was evaluated in a humanized tumor xenograft model. In this model, CD318-expressing BxPC-3 cells were subcutaneously inoculated into NCG mice for tumor formation with 5 mice per study group. Human donor PBMC were intravenously injected into the mice 5-7 days before tumor cell inoculation. The CD318-9A2 x VEGF x PDLlScFv multispecific antibody were dosed intraperitoneally at 3 mg / kg twice per week starting at day 0 post tumor cell inoculation for three weeks. The volume of the tumor and the body weight of mice were measured. The CD318-9A2 x VEGF x PDLlScFv multispecific antibody dosing led to a 50% BxPC-3 tumor cell growth inhibition compared to the PBS treatment group (Figure 12A). No differences in body weight between antibody treatment group and vehicle control group (Figure 12B). This data demonstrated the tumor inhibition efficacy of CD318-9A2 x VEGF x PDLlScFv multispecific antibody.

[0293] Example 6: Cytotoxicity of tumor cells by CD318 antibody drug conjugate (CD318-ADC)

[0294]

[0268] To explore whether CD318 can be employed as a tumor associated antigen to mediate the cytotoxicity of tumor cells, CD318 antibody drug conjugates (CD318-ADC) were made to guide the cytotoxic drug into CD318-expressing cells mediated by the CD318 antibody binding to tumor antigen. Monomethyl auristatin F (MMAF) was conjugated to CD318-9A2 VHhumlVLhuml antibody (CD318-9A2_MMAF), CD318-3A11 VHhumlVLhum2 antibody (CD318-3A11_MMAF), CD318-6662 VHhumlVLhuml antibody (CD318-6662_MMAF), an anti-HER2 antibody (HER2 MMAF), and a null control antibody (null MMAF) by oYo-Link antibody conjugation kit (AlphaThera). The efficiencies of conjugation by photo-crosslinking were verified by evaluating the conjugated products and unconjugated antibodies using SDS- PAGE under reduced conditions. The heavy chain bands of the conjugated antibodies demonstrated higher shifts in molecular weights relative to the unconjugated heavy chains due to the conjugation of two MMAF cytotoxic drug molecules while the light chain bands molecular weight sizes were unchanged (Figure 13).

[0295]

[0269] The cytotoxicity of CD318-expressing tumor cells by CD318-ADC were assessed by cell cytotoxicity assays. In these assays, increasing amounts of CD318-ADC conjugates were incubated with CD318 expressing tumor cells. The levels of cell cytotoxicity were assessed by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The CD318- 3A11_MMAF and CD318-6662_MMAF demonstrated potent cell cytotoxicity when compared to the null-MMAF (Figure 14A to 141). In most cases, the CD318-3A11_MMAF and CD318- 6662_MMAF showed similar potencies and higher levels of cytotoxicity than HER2_MMAF which correlated to the respective levels of expression of CD318 and HER2 on the tumor cells (Figure 6).

[0296]

[0270] Besides, the abilities of CD318-9A2_MMAF and CD318-6662_MMAF in the cytotoxicity of CD318-expressing tumor cells were also compared. The CD318-9A2_MMAF was less potent in cell cytotoxicity since the CD318-9A2 antibody had reduced binding affinity to CD318 when compared to CD318-6662 (Figure 15A and 15B).

[0297]

[0271] The effects of IgGl subtypes on the cytotoxicity activity of CD318- 6662_MMAF were also evaluated. CD318-6662 antibodies with either native IgGl Fc (CD318- 6662-IgGl) or IgGl Fc with L234A / L235A mutations (CD318-6662-IgGlAA) were conjugated with MMAF and evaluated in cell cytotoxicity assays with CD318-expressing tumor cells. Both CD318-6662-IgGl-MMAF and CD318-6662-IgGlAA-MMAF showed comparable potencies in the cytotoxicity of CD318-expressing cells (Figure 16A-16C), indicating that the Fc-silencing mutations did not affect the cell cytotoxicity ability of CD318-ADC molecules.

[0298] Example 7: Tumor cytotoxicity by CD318 antibody drug conjugate (CD318-ADC) in mouse tumor xenograft models

[0299]

[0272] To explore whether CD318-ADC can mediate the cytotoxicity of tumor cells in mouse tumor xenograft models, exemplary CD318 antibodies were conjugated with monomethyl auristatin E (MMAE). Briefly, CD318-9A2 VHhumlVLhuml antibody with IgGl Fc with L234A / L235A mutations (abbreviated as CD318-9A2-IgGlAA) and CD318-3A11 VHhumlVLhum2 antibody with IgGl Fc with L234A / L235A mutations (abbreviated as CD318- 3Al l-IgGlAA) were reduced in the presence of TCEP then reacted with MC-VC-PAB-MMAE (MedChemExpress) for cysteine-based cytotoxic drug conjugation. The unreacted free drugs were removed by desalting columns and the CD318-MMAE samples were concentrated by ultrafiltration. Hydrophobic interaction chromatography (HIC) was employed to analyze the CD318-MMAE molecules to estimate the drug antibody ratio (DAR). Multiple chromatographic peaks corresponding to antibodies conjugated with different numbers of MMAE were observed (Figures 17A and 17C). It was estimated that the average DAR for CD318-9A2_MMAE was 3.9 and the average DAR for CD318-3A11_MMAE was 3.5. Size exclusion chromatography (SEC) analysis of CD318-MMAE molecules revealed that both CD318-9A2_MMAE and CD318- 3A11_MMAE migrated as a single chromatographic peak without minor peaks as signs for protein aggregations (Figure 17B and 17D).

[0273] The presence of CD318 in human cell line derived xenograft tumors were characterized by immunohistochemistry (IHC) staining of tumor slice samples with anti-CD318 antibody. It was observed that CD318 had relatively high level stainings in exemplary cell line derived tumor samples from pancreatic cancer, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), gastnc cancer, colon cancer and prostate cancer (Figure 18).

[0300]

[0274] The efficacy of the CD318- ADC in tumor cell killing were evaluated in BxPC-3 human pancreatic cancer xenograft model. BxPC-3 cells were inoculated in Balb / c nude mice for the establishment of BxPC-3 tumor and testing antibody drug conjugates were intravenously administered to the mice (6 mice per group) at 5 mg / kg twice per week for two weeks. Both CD318-9A2_MMAE and CD318-3A11_MMAE led to significant tumor shrinkage with 88% and 71% tumor growth inhibitions observed for CD318-9A2_MMAE and CD318-3A11_MMAE respectively at day 38 (Figure 19A). One out of six mice treated with CD318-9A2_MMAE achieved complete tumor remission. Mice in all treated groups maintained normal body weights throughout the treatment period (Figure 19B).

[0301]

[0275] The efficacy of the CD318- ADC in tumor cell killing were also evaluated in MDA-MB-231 human triple negative cancer xenograft model. MDA-MB-231 cells were inoculated in Balb / c nude mice for the establishment of MDA-MB-231 tumor and testing antibody drug conjugates were intravenously administered to the mice (5 mice per group) at 3 mg / kg on Day 0 and Day 7. Both CD318-9A2_MMAE and CD318-3A11_MMAE led to significant tumor shrinkage with 81% and 90% tumor growth inhibitions observed for CD318- 9A2 MMAE and CD318-3A11_MMAE respectively at day 35 (Figure 19C). DS-8201 only achieved 62% tumor growth inhibition at the same dosing level. Mice in all treated groups maintained normal body weights throughout the treatment period (Figure 19D). Besides the animal study, the cytotoxicity of CD318-ADC and DS-8201 on MDA-MB-231 cells were also studied. CD318-3A11_MMAE potently killed MDA-MB-231 cells while minimal cytotoxicity activity was observed for DS-8201 in an in vitro cell cytotoxicity assay (Figure 14J).

[0302]

[0276] The efficacy of the CD318- ADC in tumor cell killing were also evaluated in HCC827 human non-small cell lung cancer xenograft model. HCC827 cells were inoculated in Balb / c nude mice for the establishment of HCC827 tumor and testing antibody drug conjugates were intravenously administered to the mice (5 mice per group) at 3 mg / kg on Day 0, 7, 21 and 36. Both CD318-9A2_MMAE and CD318-3A11_MMAE led to significant tumor shrinkage with 58% and 66% tumor growth inhibitions observed for CD318-9A2_MMAE and CD318- 3A11_MMAE respectively at day 35 (Figure 19E). DS-8201 achieved 90% tumor growth inhibition at the same dosing level. Mice in all treated groups maintained normal body weights throughout the treatment period (Figure 19F).

[0277] The efficacy of the CD318- ADC in tumor cell killing were also evaluated in RKO human colon cancer xenograft model. RKO cells were inoculated in Balb / c nude mice for the establishment of RKO tumor and CD318-3A11 MMAE was intravenously administered to the mice (5 mice per group) at 3 mg / kg on Day 1, 6, 20 and 29. CD318-3A11_MMAE led to significant tumor shrinkage with 76% tumor growth inhibition observed at day 20 compared to PBS-treated group (Figure 19G). One out of five mice treated with CD318-3A11_MMAE achieved complete tumor remission. Mice in all treated groups maintained normal body weights throughout the treatment period (Figure 19H).

[0303] Example 8: Toxicity of CD318 antibody drug conjugate (CD318-ADC)

[0304]

[0278] To explore whether CD318-ADC has any toxicity effect on normal tissues, mice were intravenously dosed with high level (17.3 mg / kg) of CD318-9A2_MMAE since CD318- 9A2 antibody can cross-react with murine CD318. Two days after dosing, mice treated with CD318-9A2_MMAE maintained similar body weights as mice dosed with PBS or null_MMAE (Figure 20A). Besides, normal levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed (Figure 20B and 20C), indicating that CD318- 9A2_MMAE treatment did not cause liver toxicity. The serum creatinine (CREA-S) and Creatine kinase-MB (CK-MB) levels were also normal (Figure 20D and 20E), indicating that CD318-9A2_MMAE treatment did not cause kidney toxicity. Complete blood count indicated that CD318-9A2_MMAE treatment did not alter the numbers of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), monocyte (Mon) or eosinophil (Eos) (Figure 20F). The counts of red blood cell (RBC) and the levels of hemoglobin (HGB) and platelet (PLT) were not affected either by CD318-9A2_MMAE treatment (Figure 20G, 20H, 201).

[0305]

[0279] Provided herein is a representative list of certain sequences included in embodiments provided herein.

[0306] Table 4 Sequences

[0307] References

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Claims

We Claim:

1. An anti-CD318 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising complementarity determining region (HCDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises SEQ ID NOs: 7, 13, or 19; the HCDR2 comprises SEQ ID NOs: 8, 14, or 20; and the HCDR3 comprises SEQ ID NOs: 9, 15, or 21; and / or a light chain variable region comprising complementarity determining region (LCDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises SEQ ID NOs: 10, 16, or 22; the LCDR2 comprises SEQ ID NOs: 11, 17, or 23; and the LCDR3 comprises SEQ ID NOs: 12, 18, or 24.

2. The anti-CD318 antibody or antigen-binding fragment of claim 1, wherein the HCDR1, HCDR2, and HCDR3 respectively comprise:SEQ ID NOs: 7, 8, and 9; orSEQ ID NOs: 13, 14, and 15; orSEQ ID NOs: 19, 20, and 21; the LCDR1, LCDR2, and LCDR3 respectively comprise:SEQ ID NOs: 10, 11, and 12; orSEQ ID NOs: 16, 17, and 18; orSEQ ID NOs: 22, 23, and 24.

3. The anti-CD318 antibody or antigen-binding fragment of any one of claims 1-2, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 101, 104, 108, and 109 or comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 101, 104, 108, and 109; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 102, 103, 105, 106, 107, 110, and 111 or comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 102, 103, 105, 106, 107, 110, and 111.

4. The anti-CD318 antibody or antigen-binding fragment of any one of claims 1-2, comprising:heavy chain variable region SEQ ID NO: 101 and light chain variable region SEQ ID NO: 102; heavy chain variable region SEQ ID NO: 101 and light chain variable region SEQ ID NO: 103; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID NO: 105; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID NO: 106; heavy chain variable region SEQ ID NO: 104 and light chain variable region SEQ ID NO: 107; heavy chain variable region SEQ ID NO: 108 and light chain variable region SEQ ID NO: 110; heavy chain variable region SEQ ID NO: 108 and light chain variable region SEQ ID NO: 111; heavy chain variable region SEQ ID NO: 109 and light chain variable region SEQ ID NO: 110; or heavy chain variable region SEQ ID NO: 109 and light chain variable region SEQ ID NO: 111.

5. The anti-CD318 antibody or antigen-binding fragment of any one of claims 1-2, comprising: a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 201, 204, 208, 209, 212, 213, and 214, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NO: 201, 204, 208, 209, 212, 213, and 214, and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211.

6. An immunocytokine comprising the anti-CD318 antibody or antigen-binding fragment of any one of claims 1-5 and a native or attenuated cytokine (e g., a native or attenuated IFNa).

7. The immunocytokine of claim 6, wherein the native or attenuated IFNa comprises an amino acid sequence of SEQ ID NO: 321, 322, 323, or 324.

8. The immunocytokine of claim 6, comprising: a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 301-312, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NO: 301-312, and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211.

9. The immunocytokine of any one of claims 6-8, wherein the immunocytokine is capable of directing native or attenuated IFNa to cells expressing CD318 and inducing death of the cells directly and / or stimulating immune cells to eliminate the cells.

10. A multispecific antibody or antigen-binding fragment thereof, comprising: an anti-CD318 moiety that targets CD318, an anti-VEGF moiety that targets VEGF, and an anti-PD-Ll moiety that targets PD-L1, wherein the anti-CD318 moiety comprises the anti-CD318 antibody or antigen-binding fragment of any one of claims 1-5.

11. The multispecific antibody or antigen-binding fragment of claim 10, wherein the multispecific antibody or antigen-binding fragment comprises one or more anti-PD-Ll moiety that is an anti-PD-Ll single chain Fv (scFv).

12. The multispecific antibody or antigen-binding fragment of claim 11, further comprising an Fc region.

13. The multispecific antibody or antigen-binding fragment of claim 12, wherein the Fc region comprises a first Fc chain and a second Fc chain, and wherein the heavy chain portion of the anti-CD318 moiety is connected to the N-terminus of the first Fc chain, the heavy chain portion of the anti-VEGF moiety is connected to the N-terminus of the second Fc chain, and the one or more anti-PD-Ll moiety is connected to the C-terminus of the first Fc chain and / or the second Fc chain.

14. The multispecific antibody or antigen-binding fragment of any one of claims 10-13, wherein the anti-VEGF moiety comprises: a heavy chain variable region comprising an amino acid selected from SEQ ID NOs: 411 and 413, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 411 and 413, and a light chain variable region comprising an amino acid selected from SEQ ID NOs: 412 and 414, or comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 412 and 414.

15. The multispecific antibody or antigen-binding fragment of any one of claims 10-14, wherein the anti-PD-Ll moiety comprises an amino acid sequence selected from SEQ ID NOs: 421 and 422, or comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 421 and 422.

16. The multispecific antibody or antigen-binding fragment of claim 13, wherein the multispecific antibody or antigen-binding fragment comprises:(i) a first heavy chain fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 401, 404, and 405,(ii) a light chain comprising an amino acid sequence selected from SEQ ID NOs: 202, 203, 205, 206, 207, 210, and 211,(iii) a second heavy chain fusion protein comprising an amino acid sequence SEQ ID NO: 402, and(iv) a light chain comprising an amino acid sequence SEQ ID NO: 403.

17. The multispecific antibody or antigen-binding fragment of any one of claims 10-16, wherein the multispecific antibody or antigen-binding fragment is capable of neutralizing VEGF and blocking PD-L1 activity around CD318-expressing cells where the anti-CD318 moiety binds to the CD318-expressing cells.

18. An antibody conjugate comprising an antibody moiety conjugated to a payload, wherein the antibody moiety is the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, or the multispecific antibody or antigen-binding fragment of any one of claims 10-17.

19. The antibody conjugate of claim 18, wherein the payload is a cytotoxic drug.

20. The antibody conjugate of any one of claims 18-19, wherein antibody moiety is conjugated to the payload through a cleavable or non-cleavable chemical linker.

21. The antibody conjugate of any one of claims 18-20, wherein the antibody conjugate is capable of killing tumor cells by targeted delivery of the payload into CD318-expressing tumor cells.

22. The antibody conjugate of any one of claims 18-21, wherein the payload is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

23. A nucleic acid encoding one or more of the polypeptides of the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, or the multispecific antibody or antigen-binding fragment of any one of claims 10-17.

24. A vector comprising the nucleic acid of claim 23.

25. A cell comprising the nucleic acid of claim 23 or the vector of claim 24.

26. A method for preparing the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, or the multispecific antibody or antigen-binding fragment of any one of claims 10-17, comprising: culturing the cell of claim 25, and obtaining the antibody or antigen-binding fragment; immunocytokine; or multispecific antibody or antigen-binding fragment from the culture, optionally using controlled Fab arm exchange of culture supernatants.

27. A method of preparing the antibody conjugate of any one of claims 18-22, comprising conjugation of the payload onto the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, or the multispecific antibody or antigen-binding fragment of any one of claims 10-17, through a cleavable or non-cleavable chemical linker.

28. A pharmaceutical composition comprising the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5,the immunocytokine of any one of claims 6-9, the multispecific antibody or antigen-binding fragment of any one of claims 10-17, or the antibody conjugate of any one of claims 18-22, and a pharmaceutically acceptable agent.

29. A method for treating or preventing a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, the multispecific antibody or antigen-binding fragment of any one of claims 10-17, the antibody conjugate of any one of claims 18-22, or the pharmaceutical composition of claim 28.

30. The method for treating or preventing a cancer according to claim 29, wherein the cancer is selected from breast cancer, bladder cancer, colon cancer, gastric cancer, lung cancer, pancreatic cancer, prostate cancer, and any cancer with CD318 expression.

31. The method for treating or preventing a cancer according to claim 30, further comprising administering chemotherapy to the subject.

32. A method for treating or preventing a disease or condition associated with overexpression of CD318 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, the multispecific antibody or antigen-binding fragment of any one of claims 10-17, the antibody conjugate of any one of claims 18-22, or the pharmaceutical composition of claim 28.

33. A method for inhibiting expression of CD318 in a cell, comprising contacting the cell with the anti-CD318 antibody or antigen-binding fragment of any one of claims 1 to 5, the immunocytokine of any one of claims 6-9, the multispecific antibody or antigen-binding fragment of any one of claims 10-17, the antibody conjugate of any one of claims 18-22, or the pharmaceutical composition of claim 28.

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